Gould's  Pocket  Pronouncing 
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By  GEORGE  M.  GOULD,  A.M.,  M.D. 


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memory   concerning   any  medical   or   surgical   theme  the  book  will 
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BJL  AKISTCM'S      C  O  NX  F»  K  1SL  P  S 

A  COMPEND 

ON 

BACTERIOLOGY 

INCLUDING 

PATHOGENIC  PROTOZOA 


BY 

ROBERT  L.  P1TFIELD,  M.  D. 

PATHOLOGIST  TO  THE   GERMANTOWN  HOSPITAL;  LATE  DEMONSTRATOR  OF 
BACTERIOLOGY   AT    THE  MEDICO-CHIRURGICAL   COLLEGE,   PHILA- 
DELPHIA; VISITING  PHYSICIAN  TO  ST.   TIMOTHY'S  HOS- 
PITAL AND  CHESTNUT  HILL  HOSPITAL,   PHILA. 


FOURTH  EDITION 

WITH  4  PLATES  AND  82  OTHER 

ILLUSTRATIONS 


PHILADELPHIA 

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PRINTED    IN     U.    S.    A. 
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PREFACE 


This  little  book  was  designed  by  the  writer  to  serve  the  needs 
of  the  medical  student  preparing  for  examination,  and  for  the 
prac  itioner  of  medicine  who  desires  to  acquaint  himself  with  the 
principle  facts  of  the  rapidly  growing  science  of  bacteriology.  An 
effort  has  been  made  to  reduce  the  subject  matter  to  as  concrete 
a  form  as  possible. 

While  the  literature  of  the  subject  of  immunity  is  as  vast  almost 
as  the  rest  of  bacteriology,  yet  it  is  hoped  that  the  chapter  in  this 
book  on  immunity  gives  in  outline  the  essential  accepted  teachings 
on  the  subject. 

Minute  details  of  cultures  and  technic  are  not  given.  They 
must  be  sought  for  in  books  on  descriptive  bacteriology. 

The  author  has  drawn  very  freely  from  many  standard  text- 
books. Many  illustrations  are  from  Kolle  &  Wassermann's 
Atlas,  Park  and  Williams,  Williams,  McFarland,  Tyson's  Prac- 
tice and  Abbott. 

The  writer's  best  thanks  are  tendered  to  Dr.  Herbert  Fox  of 
the  University  of  Pennsylvania  (Pepper  Laboratory)  to  whom 
entire  credit  is  due  for  the  chapters  on  filterable  viruses;  the  re- 
arrangement of  the  chapters,  and  the  new  matter  that  has  been 
added  throughout  the  book. 

ROBERT  L.  PITFIELD. 


4 8 885 3 


TABLE  OF  CONTENTS 


CHAPTER  I 

PAGE 

THE  CLASSIFICATION,  MORPHOLOGY,  AND  THE  BIOLOGY  OF  BACTERIA.  . .       i 

CHAPTER  II 
PRODUCTS  OF  BACTERIAL  ENERGY 23 

CHAPTER  III 
INFECTION 30 

CHAPTER  IV 

IMMUNITY 46 

CHAPTER  V 

STUDY  OF  BACTERIA 95 

CHAPTER  VI 

BACTERIOLOGICAL  LABORATORY  TECHNIC 108 

CHAPTER  VII 
ANTISEPTICS  AND  DISINFECTANTS 135 

CHAPTER  VIII 

BACTERIA 143 

CHAPTER  IX 

ANIMAL  PARASITES '. 232 

CHAPTER  X 

THE  FILTERABLE  VIRUSES 255 

CHAPTER  XI 
BACTERIOLOGY  OF  WATER,  SOIL,  AIR  AND  MILK 278 

INDEX 287 

vii 


COMPEND   OF    BACTERIOLOGY 


CHAPTER  I 

THE  CLASSIFICATION,  MORPHOLOGY,  AND  THE 
BIOLOGY  OF  BACTERIA 

BACTERIA  (fission  fungi  or  schizomycetes)  may  be  defined 
as  very  minute  unicellular  vegetable  organisms,  almost  always 
devoid  of  chlorophyll,  and  generally  unbranched,  that  reproduce 
themselves  asexually  by  means  of  direct  division  or  fission,  spores, 
or  gonidia.  They  are  allied  closely  on  the  one  hand  to  the  higher 
fungi,  such  as  the  moulds,  and  on  the  other  to  the  algae.  Many 
forms  in  one  phase  of  development  closely  resemble  members  of 
other  groups,  and  it  has  always  been  difficult  to  classify  them. 
Various  botanical  classifications  have  been  employed  by  different 
bacteriologists.  The  following  one  is  based  somewhat  upon 
Migula's,  and  that  adopted  by  Lehmann  and  Neumann,  which  was 
compiled  from  the  systems  of  Flugge,  Fischer,  Ldffler,  and  Migula. 

CLASSIFICATION. — Bacteria  may  be  conveniently  divided 
into  six  families,  according  to  their  morphology  or  shape. 

I.  COCC AC EJE.— Spherical  or  spheroidal  bacteria.  Globular 
in  free  state  but  usually  seen  with  one  axis  slightly  larger. 
They  do  not  have  parallel  sides  like  the  bacilli.  To  mul- 
tiply, the  cell  divides  into  halves,  quarters,  or  eighths,  each 
of  which  grow  again  into  perfect  spheres.  Endospores 
and  flagella  are  very  rare  (Lehmann  and  Neumann).  If 
mobile  they  are  called  Planococcus  or  Planosarcina. 


2  .  .  .  BACTERIA 

(a)  Streptococcus. — Cells  that  divic^e  in  one  direction  only 

and  grow  in  chains. 

(b)  Micrococcus. — Cells  that  divide  in  two  directions,  or 

irregularly;  with  this  group  staphylococcus  may  be 
classed.  Also  tetrads,  which  form  into  fours  by  division 
in  two  directions. 

(c)  Sarcina. — Cells  that  divide  in  three  directions  so   that 
bale-like  packages,  or  blocks  of  eight  are  formed.    At 
least  one  variety  (Sarcina  agilis)  is  motile,  having  fla- 
gella.     Plates  of  cocci,  one  thick  in  the  plane,  are  called 
"  merismopedia"" 

II.  BACTERIACE^:.— ROD  bacteria  are  straight  or  slightly 
curved.  Each  cell  is  from  two  to  six  times  as  long  as  broad. 
Division  takes  place  in  one  direction  only,  and  at  right  angles 
to  the  long  axis.  Spores  may  be  produced  or  may  not. 
They  may  have  flagella,  or  may  not. 

(a)  Bacterium.- — Neumann — Have  no  endospores.    Migula 
— no  flagella. 

(b)  Bacillus. — Neumann — Have  endospores,  and  often  grow 
in  long  threads.     Migula — Flagella  present  at  any  part 
of  cell,  peritrichic  in  arrangement.  • 

(c)  Pseudomonas. — Have  endospores  very  rarely.    Flagella 
only  at  ends. 

in.  SPIRILLACE^:.— -Spiral  bacteria.  Unicellular,  more  or  less 
elongated.  Twisted  more  or  less  like  a  corkscrew.  Cells 
are  sometimes  united  in  short  chains.  Generally  very 
motile.  Spores  are  known  in  two  varieties  only. 

(a)  Spirosoma. — Rigidly  bent.     No  flagella. 

(b)  Vibrio  or  Microspira. — Cells  that  are  rigidly  bent  like 
a  comma,  and  have  always  one,  occasionally  two  polar 
flagella. 

(c)  Spirillum. — Are  long  and  spiral,  like  a  corkscrew,  are 
rigid,  and  have  a  bunch  of  polar  flagella. 


CLASSIFICATION  3 

(d)  Spirochaeta. — Cells  with  long  flexible  spiral  threads, 
without  flagella.  Some  move  by  means  of  an  undulating 
membrane.  These  have  been  thought  to  belong  to  the 
bacteria  but  those  that  move  by  an  undulating  membrane 
should  be. classified  with  the  protozoa. 

IV.  MYCOBACTERIACE^S.— Cells  as  short  or  long  filaments, 
which  are  often  cylindrical,  clavate  (club-shaped),  cuneate 
or  irregular  in  outline,  and  display  true  or  false  branching. 
Spores  are  not  formed,  but  gonidia  are.     They  have  no  fla- 
gella, and  division  takes  place  at  right  angles  to  the  long 
axis.     There  is  no  surrounding  sheath  as  in  the  next  family  (V) . 

(a)  Mycobacterium, — Cells  are  short  cylindrical  rods,  some- 
times wedge-like,  bent,  or  Y-shaped :  long  and  filamentous. 
They  exhibit  true  branching,  and  perhaps  produce  coccoid 
elements  and  gonidia,  but  no  flagella.    The  Corynebac- 
terium  of  Lehmann  and  Neumann  belongs  to  this  group. 
Many  are  acid-fast. 

(b)  Streptothrix    or   Actinomyces    (ray  fungus)    are   long 
mycelial  threads,  that  radiate  in  indian-club,  or  loop-like 
forms,  with  true  branching  and  delicate  sheaths,  devoid  of 
gonidia  and  flagella.     Growth  coherent,  mould-like  and 
dry.     Often  powdery  on  the  surface  in  culture  media, 
frequently  emitting  a  musty  odor.     A  few  species  are 
weakly  acid-fast. 

V.  CHLAMYDOBACTERIACE^:.— Sheathed  bacteria.    Cells 
are  characterized  by  an  enveloping  sheath  about  branched 
and  unbranched   threads.     Division   takes  place  at  right 
angles  to  the  long  axis  of  the  cells. 

(a)  Cladothrix  are  distinguished  by  false  dichotomous 
branching.  Multiplication  is  affected  by  separation  of 
whole  branches,  and  by  swarm  spores  or  motile  gonidia 
having  flagella. 


4  BACTERIA 

(b)  Crenothrix. — Filaments  are  fixed  to  a  nutrient  base. 
Are  usually  thinner  at  the  base  than  at  the  apex,  formed 
of  unbranched  threads  that  divide  in  three  directions  of 
space,  and  produce  in  the  end  two  kinds  of  gonidia, 
probably  of  bisexual  nature. 

(c)  Phragmidiothrix. — Cells  are  first  united  into  unbranched 
threads  by  means  of  delicate  sheaths,  branching  threads 
are  then  formed.     Division  takes  place  in  three  directions 
of    space,    producing    sarcina-like    groups    of    gonidia, 
which,  when  free,  are  spherical. 

(d)  Thiothrix. — Are    unbranched    cells,    sheathed,   without 
flagella,  divided  only  in  one  direction^  and  contain  sulphur 
granules. 

VI.  BEGGIATOACE^:.— Cells  united  to  form  threads  that  are  not 
sheathed:  have  scarcely  visible  septa;  divide  in  one  direction, 
and  motile  only  by  an  undulating  membrane,  not  by  flagella. 
(a)  Beggiatoa. — Cells  containing  sulphur  granules. 

Bacteria  may  furthermore  be  classified  according  to  their  biolog- 
ical characteristics,  which  may  be  wonderfully  different.  The  ulti- 
mate differentiation  of  one  species  from  another  depends  not  only 
on  the  morphology,  which  may  be  precisely  similar,  but  on  its  bio- 
logical behavior  in  culture  media  and  in  the  tissues  of  animals  un- 
der identical  conditions.  Again,  different  individuals  of  a  given 
species  may  vary  extraordinarily  one  from  another  in  form  and 
size,  yet  the  chemical  behavior  is  invariably  the  same.  Hence  it 
is  only  by  observation  of  the  development  of  bacteria  in  culture 
media,  and  the  reactions  produced  in  it,  and  in  the  bodies  of  ex- 
periment animals,  that  we  can  identify  them  positively  from  others 
of  a  foreign  species.  No  bacteriologist  is  able  by  a  simple  micro- 
scopical examination  of  a  given  bacterium,  to  identify  it  absolutely 
at  all  times. 

The  higher  groups  of  fungi  may  be  classified  conveniently  as 
follows : 


CLASSIFICATION  5 

BLASTOMYCETES-YEASTS.— Budding  fungi.  Character- 
istic lies  in  predominant  round  or  elliptical  unit;  some  few  form 
mycelia;  division  by  endospores  or  budding;  important  in  fer- 
mentation and  in  disease.  Divided  into: — 

Saccharomyces. — Endospores  and  budding,  fermenters.  No 
mycelia. 

Monilia. — Budding.     No  spores — mycelia — fermenters. 

Oidia. — Budding.     No  spores — mycelia — non-fermenters. 

Coccidioides. — Spores. — No  budding — mycelia — non-fermen- 
ters. 

HYPHOMYCETES-MOULDS.— Mycelium-forming  fungus; 
division  by  spores,  branching,  budding,  or  intercalary  division; 
some  bisexual.  Divided  into: — 

Phycomycetes. — Mucorinae — sometimes  bisexual — division  by 
grouped  spores  or  segmentation  of  mycelium.  Not  important 
pathologically.  Example — Mucor. 

Mycomycetes. — Asexual  forms  dividing  by  spores  in  a  sac 
or  by  end  organs — sexual  forms  dividing  by  specially  developed 
cells.  Mycelia  predominate.  Example — Aspergillus. 

These  are  the  principal  groups  of  yeasts  which  can  be  reasonably 
well  classified.  There  are  others,  Microsporon,  Trichophy  ton,  and 
Sporothrix,  that  have  a  decided  pathogenic  importance  but  for 
which  a  systematic  position  is  not  easy  to  give.  They  belong 
probably  between  the  two  above  classes  in  that  mycelial  growth 
with  lateral  budding  and  spore  formation  are  their  characters. 

Bacteria  that  are  globular  in  form  are  called  cocci. 

Cocci  that  divide  in  one  direction  of  space  and  grow  in  chains 
are  called  streptococci  (Fig.  i). 

Cocci  that  divide  irregularly  and  form  parrs  of  fours,  or  irregular 
groups,  are  called  micrococci.  Those  of  this  class  that  form  pairs 
are  frequently  called  diplococci.1  When  they  form  fours  by  divi- 
sion in  two  directions,  they  are  called  tetrads.  But  when  they 

1  This  word  is  frequently  used  as  if  it  were  a  biological  term  indicating  some 
species  identity,  e.g.,  Diplococcus  pneumoniae.  There  is  no  biological 
group  called  Diplococci  and  the  term  should  be  used  in  a  descriptive  sense. 
The  cause  of  pneumonia  is  now  called  Streptococcus  pneumoniae. 


BACTERIA 


divide  irregularly  and  form  masses  resembling  bunches  of  grapes, 
they  are  spoken  of  a  staphylococci  (Fig.  2). 

Coeci  that  divide  in  three  directions  are  called  sarcina.     One 
single  coccus,  by  division  in  three  directions,  forms  cubes  of  eight 


FIG.  i.  —  Large  and  very  large  streptococci.     (Kolle  and  Wassermann.) 

or  more,  each  of  which  becomes  globular  and  equal  in  size  to  the 
parent. 

Motile  micrococd  are  those  that  divide  in  two  directions  of  space 
and  have  flagella.     They  are  known  as  planococci. 


FIG.  2.  —  Staphylococci.     Streptococci.     Diplococci.    Tedrads.     Sarcinae. 

(Williams.) 

Micrococci  that  divide  in  three  directions,  and  are  motile,  are 
called  planosarcina  (Fig.  3). 

Bacteria  that  resemble  straight  rods  are  called  bacilli.  These 
may  be  short  and  thick,  or  long  and  thread-like;  are  never  curved, 
but  may  be  slightly  bent. 


CLASSIFICATION 


Bacilli  may  grow  singly  or  in  chains;  may  be  flagellated;  contain 
spores  and  gonidia;  or,  may  be  devoid  of  flagella. 
Members  of  the  spirillaceae  that  resemble  a  curved  rod,  or  are 


FIG.  3. — Planosarcina  ureae,  showing  very  long  flagella. 
(Kolle  and  Wassermann.) 

comma-shaped,  are  known  as  vibrios  (Fig.  4).  Those  of  the  same 
family  that  resemble  a  corkscrew,  are  called  spirilla.  When  they 
are  like  long  spiral  threads  they  are  called  SpirocJUttd  (Fig.  5) . 

Any  of    these    different  members  of   the 
family  of  Spirillaceae  may  grow  in  chains. 

In  clinical  medicine  it  is  common  to  speak 
of  the  streptococcus  pneumonia  as  the  pneu- 
mococcus.  As  the  organism  appears  in  the 
diseased  lung,  or  in  the  sputum,  one  diameter 
of  the  coccus  is  invariably  longer  than 
another,  and  the  rule  of  equal  diameters 
cannot  be  applied  to  it.  But  in  culture 
media,  the  organism  resembles  a  true  coccus, 
being  globular  and  growing  in  chains.  It  is  then  called  the 
Streptococcus  pneumoniae.  It  is  common  also  to  speak  of  mem- 
bers of  the  family  of  Mycobacteriacea  as  bacilli,  as  they  are  more 


FIG.  4. — Cholera 

vibrios. 

(Greene's  Medical 
Diagnosis.) 


8  BACTERIA 

commonly  met  with  in  this  form  in  clinical  examinations,  and  in 
cultures.  Hence,  we  frequently  hear  of  the  bacillus  of  tubercu- 
losis, and  not  the  Mycobacterium  tuberculosis. 

Among  the  higher  bacteria,  the  differentiation  of  those  belong- 
ing to  the  sheathed  group,  or  Chlamydobacteriacea,  is  difficult,  as  it 
depends  largely  upon  the  formation  of  the  false  branching  and 
the  gonidia.  When  bacteria  exhibit  many,  or  various  forms, 
in  the  same  culture,  as  does  the  typhoid  bacillus,  we  speak  of 


FIG.  5. — Spirochaeta  of  relapsing  fever.     (Kolle  and  Wassermann.) 

them  as  pleomorphic,  or  as  showing  pleomorphism.  To  eluci- 
date: Man  is  pleomorphic,  because  among  adult  individuals 
some  are  tall  or  short,  fat  or  thin. 

Involution  or  Degeneration  Forms. — When  the  best  or  opti- 
mum conditions  for  bacterial  life  (see  page  18)  are  not  found, 
bacteria  present  appearances  quite  different  from  those  of  the 
young,  active  or  perfect  adult  type.  These  changes  are  called 
involutionary  if  temporary,  or  degenerative  if  permanent. 
For  example:  the  diphtheria  bacillus  under  good  conditions  for 
life  is  a  straight  or  slightly  bent  rod  staining  in  a  granular  manner. 


CLASSIFICATION  Q 

If  living  under  unsuitable  conditions  it  becomes  quite  short, 
and  stains  solidly.  Again,  bacilli  that  are  accustomed  to  appear 
as  short  elements  may  grow  to  long  threads  without  dividing, 
or  swell  into  unrecognizable  form.  Branching  is  sometimes 
seen  in  rods  and  spirals,  a  condition  due  in  certain  cases  to 
involution,  in  others  naturally  among  the  higher  bacteria. 

To  measure  bacteria,  we  use  the  thousandth  part  of  a  milli- 
meter, called  the  micromillimeter,  or  micron,  as  the  unit.  The 
Greek  letter  ju  is  the  symbol  for  this  unit.  A  micron  is  about 
M5>ooo  of  an  inch,  yet  a  bacterium  i  n  long,  and  %  fj.  in  width, 
is  very  large  in  comparison  to  some  things  that  scientists  measure, 
such  as  the  thickness  of  oil  films,  soap  bubbles,  or  light-wave 
lengths,  in  which  the  unit  is  a  micromicron,  and  is  symbolized 
by  /*/*.  The  shortest  light-wave  lengths  are  about  400  /*/*>  or 
.4  ju,  while  chromatic  threads  in  cells  of  bacteria  are  often  100  w 
in  width.  Then  again  there  are  many  things  smaller  than  these 
threads.  The  thinnest  part  of  a  bursting  soap  bubble  is  but 
7  juju  in  thickness.  There  are  certain  infectious  agents  that  are 
submicroscopic;  that  is,  invisible  even  by  the  aid  of  Siedentopf's 
ultraviolet  microscope,  which  shows  objects  smaller  by  half  a 
light- wave  length  (.2  AIJU). 

The  structure  of  the  bacterial  cell  is  very  simple,  consisting  of 
a  delicate  poorly  staining  limiting  membrane  or  wall  enclosing 
a  mass  of  substance  with  strong  affinity  for  basic  dyes  like 
methylene  blue.  Just  what  part  of  the  bacterial  interior  is 
cytoplasm  and  what  is  nucleus  is  not  definitely  known.  Some 
observers  believe  that  all  that  is  stained  is  chromatin,  or  nuclear 
matter  diffusely  distributed  through  the  bacterial  cell,  while 
others  think  that  a  delicate  cytoplasm  exists  under  the  wall  and 
that  it  is  overshadowed  by  relatively  great  proportional  bulk 
of  the  nucleus. 

In  the  course  of  the  rod  we  often  see  metachromatic  bodies, 
called  the  Babes-Ernst  granules,  and  unstained  spaces  called 
vacuoles,  both  of  which  are  common  to  many  bacteria.  They 


10  BACTERIA 

may  be  ingested  substances  but  some  are  lipoidal  or  carbohy- 
drate in  nature.  These  bodies  are  demonstrated  by  staining 
with  basic  dyes  and  may  be  of  importance  in  determining  the 
mycobacteria.  It  is  thought  that  they  play  a  role  in  reproduc- 
tion (Fig.  65). 

The  food  of  the  bacterium  passes  through  the  cell  wall  by 
osmosis.     The  cell  wall  of  certain  organisms,  for  example  the 


FIG.  6. — Zooglea  formation.     (Leuconostoc.)     (Kolle  and  Wassermann.) 

pneumococcus,  undergoes  a  change  whereby  a  mucilaginous  or 
gelatinous  capsule  is  formed  outside  the  cell  wall.  Its  use  is 
not  known.  The  cell  wall  is  generally  the  first  portion  of  the  cell 
to  be  attacked  by  certain  specific  substances  (ferment)  found 
in  the  blood  of  immunized  animals,  called  bacteriolysins  and 
agglutinins.  Where  great  masses  of  bacteria  are  clumped  in 
excessive  mucilaginous  material  we  speak  of  this  condition  as 
zooglea  (Fig.  6). 

We  sometimes  find,  as  a  prolongation  of  the  cell  wall,  filament- 
ous organs  of  locomotion  known  as  flagella.  Bacteria  without 
flagella  are  sometimes  called  gymnobacteria,  those  possessing 


CLASSIFICATION 


II 


them,  trichobacteria  but  these  terms  are  falling  into  disuse 
because  the  latter  is  now-a-days  applied  to  higher  groups  that  grow 
in  hair  like  forms.  However  the  following  may  be  described: 
When  they  have  one  flagellum  we  call  them  monotrichous  bacteria, 
and  amphitrichous  when  there  are  two  flagella,  one  at  each  pole 
(Fig.  7).  When  the  cell  is  surrounded  by  flagella,  it  is  known  as 
a  peritrichous  bacterium,  and  lopho- 
trichous  when  the  flagella  are  ar- 
ranged in  tufts  of  two  or  more. 
These  are  simple  adjectives  and  not 


FIG.  7. — Spirillum  undula  with  polar 
flagella.     (Kolle  and  Wassermann.) 


FIG.  8. — Bacillus  proteus  vul- 
garis,  showing  peritrichous  fla- 
gella. (Kolle  and  Wassermann.) 


now  used  as  terms  of  classification.  The  tetanus  bacillus  is  an 
example  of  a  peritrichous  organism,  while  the  bacillus  of  green 
pus  is  called  monotrichous,  because  of  its  single  flagellum. 

Flagella  are  not  pseudopods,  but  distinct  organs  of  locomo- 
tion. 

In  certain  bacteria  of  the  Beggiatoa,  locomotion  is  accom- 
plished by  a  peculiar  amoeboid  motion,  or  by  an  undulating 
membrane.  On  looking  at  bacteria  known  to  have  no  powers 


12  BACTERIA 

t 

of  voluntary  motion,  they  are  seen  to  oscillate,  tremble  or  move 
slightly.  Suspensions  of  india-ink  in  water  are  seen  to  do  the 
same  thing,  as  are  other  inanimate  suspensions.  This  molecular 
movement  is  known  as  the  Brownian  motion.  By  ordinary 
staining  methods,  and  in  preparations  of  living  bacteria  known 
to  be  flagellated,  these  organs  of  locomotion  cannot  be  seen, 
as  a  rule.  Occasionally,  however,  one  may  be  seen  under  either 
condition.  Generally,  strong  solutions  of  aniline  dyes,  to  which 
powerful  mordants  have  been  added,  are  necessary  to  stain  the 
capsule  of  bacteria  and  the  attached  flagella.  The  motion  of 
bacteria  varies  from  a  simple  rotatory,  on  one  axis,  to  a  swing- 
ing, shaking,  boring  or  serpentine  action.  The  location  of  the 
flagella  has  some  influence  upon  the  motion  they  impart. 
Flagella  may  be  broken  off  from  the  cell  body  by  agitation, 
but  when  separated  may  still  be  clumped  by  agglutinating  sera. 

Flagella  may  have  other  functions  than  locomotion.  It  is 
possible  that  they  serve  as  organs  for  the  absorption  of  nour- 
ishment from  the  surrounding  media.  The  presence  of  very  long 
or  very  numerous  flagella  does  not  necessarily  presage  very 
active  motion.  At  times,  under  certain  conditions,  an  organism 
ordinarily  motile  and  flagellated  will  appear  immobile  and  non- 
flagellated  (Lehmann  and  Ziferler),  but  this  is  rare.  Certain 
flagella  have  in  their  continuity  little  round  granules,  or  bodies, 
which  apparently  have  nothing  to  do  with  the  functions  of 
locomotion  but  may  have  something  to  do  with  the  nutrition 
of  the  cell.  The  test  of  motility  of  a  bacterium  is  to  see  it  pro- 
gress by  itself  completely  across  the  field  of  the  microscope. 

REPRODUCTION.— The  process  of  direct  cell  division  is 
the  commonest  way  by  which  bacteria  multiply;  hence  comes 
the  name  of  fission  fungi.  The  ways  of  reproduction  of  the 
bacteria  high  in  the  scale  are  by  direct  division,  branching,  and 
by  means  of  spores,  and  by  other  granules  called  gonidia.  The 
spores  appearing  in  the  lower  bacteria,  bacilli  for  example,  are 
not  reproduction  forms  but  states  of  high  resistance. 


SPORULATION  13 

The  process  of  direct  or  binary  division  is  very  simple,  and 
may  be  a  matter  of  twenty  minutes,  or  as  long  as  six  hours. 
Division  is  almost  always  across  the  cell  in  the  direction  of  the 
short  axis,  though  it  may  in  some  bacteria  be  in  a  direction 
parallel  to  the  long  axis,  but  this  is  uncommon. 

By  means  of  the  hanging-drop  or  the  block-culture  method, 
on  an  inverted  cover-glass  the  process  may  be  observed  easily. 
The  phenomena  of  division  begin  by  an  elongation  of  the  cell, 
soon  followed  by  a  constriction  of  pinching  in  of  the  cell  on  both 
sides,  at  an  equatorial  point.  The  process  begins  to  be  apparent 
in  the  cell  wall  and  extends  inward. 

Division  may  occur  in  one,  two,  or  three  directions,  or  planes. 

By  cell  division  bacteria  multiply  by  geometrical  progression. 
One  cell  at  the  end  of  a  period  becomes  two,  and  at  the  end  of  a 
second  period  these  two  become  four;  at  the  end  of  another 
period  these  four  become  eight;  after  twenty-four  hours  they 
may  number  many  millions. 

It  is  well  that  the  food  supply  soon  gives  out  and  that  the 
products  of  bacterial  metabolism,  such  as  acids  and  ferments, 
inhibit  their  growth.  By  this  rapid  bacterial  multiplication, 
carcasses  of  animals  are  disintegrated  and  the  higher  nitrogenous 
compounds  are  reduced  to  simple  gases  that  are  quickly  dissi- 
pated in  the  air. 

SPORULATION.— Sporulation  is  of  two  kinds:  the  first  and 
most  important  for  hygiene  is  that  into  which  some  pathogenic 
bacteria  go  when  they  meet  unfavorable  conditions  and  it  affords 
protection  against  all  but  the  most  vigorous  disinfection;  the 
second  kind  is  a  specialized  function  of  the  higher  bacteria  and 
moulds  by  which  reproduction  occurs  (vegetative).  In  the 
latter  case  it  is  not  impossible  that  some  sexual  specialization 
occurs.  The  first  mentioned  are  called  Endospores. 

Vegetative  sporulation  corresponds  to  the  flowering  of  the 
higher  plants,  and  is  observed  under  the  most  favorable  vital 
conditions.  Endospores  are  produced  under  stress  of  circum- 


BACTERIA 


stances,  when  certain  agencies  or  conditions,  such  as  absence  of 
food,  drying,  and  heat,  threaten  the  extinguishment  of  the  organ- 
ism. Spores  are  bright,  shining,  oval,  or  round  bodies,  which  do 


PIG.  9. — The  formation  of 
spores.  (After  Fischer  from 
Frost  and  McCampbell.) 


FIG.  10. — Spores  and  their  location  in  bac- 
terial cells.     (After  Frost  and  McCampbell.) 


not  take  aniline  dyes  readily,  and  which,  when  they  are  stained, 
retain  the  color  more  tenaciously  than  the  adult  cells.  They 
resist  heat,  often  withstanding  a  temperature  of  i5o°C.  dry  heat 
for  an  hour.  Steam  under  pressure  at  a  temperature  of  i5o°C. 
will  invariably  kill  them  after  a  short  exposure. 


0   0  fl  8 


FIG.  ii. — Spore  germination,  a,  direct  conversion  of  a  spore  into  a  bacillus 
without  the  shedding  of  a  spore-wall  (B.  leptosporus) ;  b,  polar  germination  of 
Bad.  anthracis;  c,  equatorial  germination  of  B.  suUilis;  d,  same  of  B.  mega- 
terium;  e,  same  with  "horse-shoe"  presentation.  (After  Novy.) 

Spores  are  situated  either  in  the  ends  of  the  adult  organism 
(polar)  or  in  the  middle  (equatorial),  and  the  spore  is  discharged 
(sporulation)  either  from  the  end  or  through  the  side. 


SPORULATION  .          15 

The  spore  is  developed  in  the  bacterial  cell  as  follows:  If 
the  organism  is  a  mobile  one  it  becomes  quiet  before  sporulation, 
during  which  the  flagella  are  retained.  The  position  of  the 
spore  is  early  marked  by  a  granularity  of  the  bacterial  body  at 
one  point,  an  area  soon  assuming  a  clear  glistening  character, 
often  with  a  double  contour,  which  may  or  may  not  increase  the 
thickness  of  the  cell.  If  unfavorable  conditions  continue  the 
cell  body  disintegrates  and  disappears  leaving  the  spore  bare. 


?IG.  12. — Capsules.     Bad.  pneumonia  (Friedlander;.     (After  Weichselbaum 
from  Frost  and  McCampbell.) 

Certain  spore  bearing  bacteria  grown  for  a  week  at  42°C.  lose 
the  power  to  form  spores;  likewise  their  progeny.  As  a  rule  the 
anthrax  bacillus  does  not  form  spores  in  the  bodies  of  animals. 
Free  oxygen  is  required  for  sporulation  by  some  bacteria.  One 
spore  only  is  produced  by  an  adult  cell.  Some  forms  of  bacteria 
can  be  differentiated  from  each  other  only  by  the  way  in  which 
they  sporulate,  whether  from  the  poles  or  the  equator. 

Spores  are  formed  chiefly  by  the  rod-shaped  bacteria  especially 
the  anaerobic  and  saprophytic  organisms  and  these  varieties 
always  have  a  high  thermal  death-point.  Certain  round  bodies 


I 6          .  BACTERIA 

found  in  bacteria  of  high  thermal  death-point,  are  called  by 
Heuppe  arthrospores.  It  is  believed  that  they  are  without 
significance.  Arthrospores  are  common  among  the  micrococci 
and  may  be  associated  with  capsule  formation  and  cell  enlarge- 
ment. The  whole  cell  may  stain  more  intensely.  They  are  also 
to  be  sought  among  the  Streptothrix  genus. 

Spores  resist  chemicals  for  a  long  period,  and  withstand  drying, 
even  in  lime  plaster,  for  years.  It  is  believed  that  the  thick 
capsule  enables  them  to  resist  these  deleterious  agents. 


FIG.  13. — Pest  bacilli  showing  capsules.     (Kolle  and  Wassermann.) 

Sporulation  is  more  apt  to  occur  under  poor  nutritive  conditions. 

The  anthrax  bacillus  thrives  at  i3°C.  but  cannot  sporulate 
below  i8°C.  Anthrax  spores  have  been  known  to  resist  the 
germicidal  action  of  a  5  percent  carbolic  acid  solution  for  forty 
days. 

Capsules. — Certain  well-known  pathogenic  bacteria  have 
thick  well-marked  capsules.  The  pneumococcus,  pneumobacillus, 
and  Bacillus  aerogenes  capsulatus,  are  well-known  examples  of 
such  capsulated  organisms.  The  capsule  is  not  always  constant. 
It  often  disappears  when  the  organism  is  grown  in  culture 
media  (Figs.  12  and  13). 


THE   CHEMICAL   COMPOSITION   OF  BACTERIA  17 

The  higher  bacteria  are  those  from  the  Mycobacteriacea  up  to 
the  yeasts  and  moulds.  They  are  higher  than  the  Bacteriacea 
because  they  tend  to  form  truly  or  falsely  branching  filaments 
and  specialized  segments,  gonidia,  which  may  behave  as  sex 
organs.  Few  of  them  are  pathogenic,  except  in  the  genera 
Mycobacterium  and  Streptothrix.  To  the  former  belongs  the 
diphtheria  and  tubercle  bacillus,  both  of  which  are  said  to  have 
branching  involution  forms,  while  to  the  latter  belong  the  organ- 
isms of  actinomycosis  and  Madura  foot.  The  Chlamydo- 
bacteriacea  and  Beggiatoa  are  Saprophytes.  These  require  special 
technique  for  the  laboratory  culture. 

The  Yeasts  or  Blastomycetes  or  budding  fungi  are  next  in  order. 
They  consist  of  sharply  and  doubly  outlined,  refractive,  oval 
bodies  which  may  grow  out  into  short  stalks  called  mycelia.  They 
grow  well  in  the  laboratory  and  may  produce  pigments.  They 
are  much  larger  than  the  bacteria  (10-25  /x  long).  They  multi- 
ply by  budding  with  a  separation  and  removed  growth  of  the 
young  form.  They  may  produce  a  local  or  general  infection  in 
man,  Blastomycosis.  They  are  used  in  beer  making.  The 
commonest  genus  is  Saccharomyces. 

The  Moulds  or  Uyphomycetes  represent  the  next  highest  group 
of  the  plant  algae.  They  are  characterized  by  a  greater  promi- 
nence of  the  mycelium  over  simple  segments  or  bodies.  They 
are  widespread  in  nature  and  many  are  pathogenic.  They 
multiply  by  segmentation  of  the  mycelia  into  gonidia  or  by  the 
development  of  special  spore  masses  called  sporangia.  Fur- 
ther refinements  of  the  spores  into  sexual  elements  is  known. 
They  are  chiefly  of  interest  to  the  physician  on  account  of  the 
skin  diseases  that  they  occasion. 

THE  CHEMICAL  COMPOSITION  OF  BACTERIA 

Bodies  of  bacteria  contain  water,  salts,  certain  albumins,  and 
bodies  that  may  be  extracted  with  ether.  Among  the  latter  are 
lecithin,  cholesterin,  and  triolein.  In  acid-fast  organisms,  fatty 


1 8  BACTERIA 

acids  and  wax  have  been  found.  In  others,  xanthin  bases,  cellu- 
lose, starch,  chitin,  iron  salts,  and  sulphur  grains  have  been  dis- 
covered. The  essential  protein  of  the  cell  body  is  highly  nitrog- 
enous and  is  usually  combined  with  some  carbohydrate  as  a 
glyconucleo-protein.  The  salts  in  the  ash  are  mostly  composed 
of  various  phosphates.  Intracellular  toxins  in  combination 
with  the  cytoplasm  are  found  in  certain  groups  of  bacteria, 
e.g.,  B.  typhosus. 

BIOLOGICAL  CONDITIONS 

Bacteria  are  arbitrarily  classes  as  parasites,  or  saprophytes. 
They  may  be  so  dependent  upon  the  tissues  of  the  infected 
organism  as  to  be  a  strict  parasite  and  incapable  of  growth  under 
any  other  condition  (Mycobact.  leprce),  or  they  may  be  capable 
of  life  on  artifical  culture  media  (tubercle  bacillus),  or  of  life  in 
the  body,  on  culture  media  containing  organic  matter  (influenza 
bacillus),  or  in  the  soil  (B.  tetani). 

Saprophytes  are  bacteria  capable  of  living  upon  dead  organic 
matter,  in  soil,  in  water,  in  air;  they  are  not  parasitic  and  do  not 
resist  the  defenses  of  the  living  body. 

Certain  biological  conditions  are  essential  for  the  growth  of 
bacteria:  water,  oxygen,  carbon;  nitrogen,  and  salts  are  neces- 
sary. For  certain  parasitic  bacteria,  highly  complex  substances 
are  indispensable:  meat  albumins,  peptones,  milk,  egg  albumin, 
blood  serum,  and  sugars  are  the  ingredients  of  various  culture 
media. 

The  chemical  reaction  of  such  media  is  important:  it  should 
either 'be  faintly  acid  or  faintly  alkaline.  The  greatest  number 
of  water  bacteria  grow  in  media  that  are  slightly  acid,  while 
diphtheria  produces  its  strongest  toxins  and  grows  best  in 
alkaline  media.  Salt-free  media  is  required  for  a  number  of 
pathogenic  bacteria,  e.g.,  the  gonococcus,  B.  leprae. 

All  bacteria  require  for  their  growth  either  free  oxygen,  as  in 
air,  or  combined  oxygen,  as  in  albumin,  water,  etc.  Those  that 


BIOLOGICAL    CONDITIONS  1 9 

only  grow  when  deprived  of  free  oxygen  are  known  as  obligate 
anaerobes,  while  those  that  require  the  presence  of  oxygen  are 
called  obligate  aerobes.  Those  that  grow  under  either  conditions 
are  named  facultative  anaerobes.  Free  oxygen  is  needed  for  spore 
formation  by  certain  bacteria.  Anaerobes  obtain  oxygen  as 
they  need  it  by  breaking  up  their  foodstuffs. 

Nutriment  is  most  important  for  the  growth  of  bacteria, 
nitrogenous  compounds  (albumins)  particularly  being  required. 
Simple  aquatic  forms  of  bacteria  can  live  and  grow  in  distilled 
water.  The  addition  of  the  various  sugars  is  of  advantage  in 
the  cultivation  of  many  bacteria,  and  glycerine  for  the  growth 
of  some  members  of  the  Mycobacteriacea.  Blood  serum  or  whole 
blood  is  required  by  some  pathogenic  organisms.  The  foodstuffs 
must  be  in  a  form  that  can  diffuse  through  the  cell  wall. 

The  temperature  of  the  medium  in  which  various  bacteria 
grow  is  most  important.  Bacterial  growth  is  possible  between 
o°C.  and  7o°C.,  some  varieties  thrive  at  the  one  extreme,  and 
others  at  the  other. 

Psychrophilic  bacteria,  are  those  that  grow  at  i5°C.,  with  a 
maximum  of  3o°C.  and  a  minimum  of  o°C.  Water  bacteria  of 
the  polar  seas  belong  to  this  group. 

Mesophilic  grow  best  at  37°C. — the  temperature  of  the  body— 
and  thrive  from  io°C.  (minimum)  to  45°C.  (maximum).  All 
pathogenic  bacteria  belong  to  this  group. 

Thermophilic  (min.  temp.  4o°C.,  max.  6o-7o°C.)  are  most 
prolific  at  50-5  5 °C.  To  this  class  belong  bacteria  of  the  soil. 
All  of  this  class  are  spore-bearing. 

Darkness  favors  bacterial  growth. 

Association  of  different  kinds  of  bacteria  is  of  some  importance 
in  their  growth  and  welfare  and  when  thus  associated,  they  some- 
times benefit  each  other.  Such  combination  is  called  symbiosis. 
Antibiosis  is  the  condition  when  one  or  more  of  a  mixture  of 
organisms  suffers  by  the  presence  of  others,  e.g.,  the  destruction 
of  putrefactive  germs  in  the  intestinal  tract  by  lactic  acid  bacilli. 


20  BACTERIA 

Certain  anaerobic  bacteria  grow  in  the  presence  of  oxygen  if 
other  particular  varieties  of  aerobic  bacteria  are  present. 

Attenuated  tetanus  bacilli  become  virulent  if  cultivated  with 
Bacterium  vulgae.  Again,  complicated  chemical  changes,  as  the 
decomposition  of  nitrites  with  the  evolution  of  nitrogen  cannot 
be  accomplished  by  certain  bacteria  severally,  but  jointly,  this 
is  quickly  brought  about. 

Pfeiffer  has  shown  that  certain  chemical  substances  (foods, 
albumins,  etc.),  attract  bacteria  (positive  chemotaxis) ,  while 
other  substances,  as  turpentine,  repel  them  (negative  chemotaxis). 
Oxygen  repels  anaerobes  and  is  particularly  attractive  to  aerobes. 

FREE  AGENTS  PREJUDICIAL  TO  THE  LIFE  OF 
BACTERIA 

High  temperatures  are  surely  germicidal:  6o°C.  coagulates 
mycoprotein  of  bacteria  and  other  common  albumins.  The  degree 
of  temperature  at  which  bacteria  are  killed  is  called  the  thermal 
death-point.  Most  vegetative  forms  die  after  a  short  exposure  at 
6o°C.,  though  some  require  a  higher  temperature,  e.g.,  tubercle 
bacillus. 

Spores  resist  boiling,  often  for  hours.  Spore-bearing  bacilli 
from  the  soil  often  survive  a  temperature  of  ii5°C.  moist  heat 
(steam),  from  thirty  to  sixty  minutes.  Bacteria  resist  dry  heat 
of  i75°C.  from  five  to  ten  minutes. 

Cold  inhibits  bacteria;  destroys  some;  but  is  not  a  safe  germi- 
cidal agent,  as  typhoid  bacilli  have  been  isolated  from  melted  ice 
in  which  they  had  been  frozen  for  months. 

Ravenel  exposed  bacteria  to  the  extreme  cold  of  liquid  air 
(— 3i2°F.)  and  found  that  typhoid  bacilli  survived  an  exposure 
of  sixty  minutes;  diphtheria,  thirty  minutes,  and  anthrax  spores, 
three  hours;  during  this  exposure,  however,  many  were  destroyed. 

Light  is  inimical  to  the  life  of  bacteria,  direct  sunlight  being  the 
most  germicidal,  as  it  destroys  some,  reduces  the  virulence  of 


AGENTS   PREJUDICIAL   TO  BACTERIAL   LITE  21 

others,  or  interferes  with  the  chromogenic  properties.  Typhoid, 
cholera,  diphtheria,  and  many  other  organisms  are  killed  after  an 
hour  or  two's  exposure  to  bright  sunlight.  The  ultraviolet  or 
actinic  rays  are  the  efficient  ones.  If  free  oxygen  is  excluded,  the 
germicidal  action  is  very  materially  reduced.  Sunlight  acting 
on  culture  media  (free  oxygen  and  water  being  present)  produces 
after  ten  minutes,  peroxide  of  hydrogen.  This  action  of  light  on 
bacteria  has  been  extensively  used,  notably  by  Hansen,  as  a 
therapeutic  measure  for  the  cure  of  bacterial  skin  diseases,  espe- 
cially lupus.  Diffuse  sunlight,  electric  light,  Rcentgen-rays,  con- 
tinuous and  alternating  currents  of  electricity,  are  also  more  or 
less  germicidal.  Antiseptics,  such  as  metallic  salts,  formalin, 
carbolic  acid,  cresol,  mineral  acids,  and  essential  oils,  are  powerful 
germicides;  some  even  in  high  dilution. 

According  to  Koch,  absolute  alcohol,  glycerine,  distilled  water, 
and  concentrated  sodium  chloride  solution  do  not  affect  anthrax 
spores,  even  after  acting  on  them  for  months.  Halogen  elements 
(iodine,  bromine,  chlorine)  are  the  most  powerful  germicides. 

Free  acids  and  alkalies  must  be  very  strong  to  act  as  disin- 
fectants. Excessive  amounts  of  sugar,  salt,  glycerine,  and  the 
pyroligneous  acids  act  as  destroyers,  or  inhibitors  to  bacterial 
growth  in  foodstuffs. 

Metals  act  as  lethal  agents  in  the  presence  of  light  and  water,  by 
forming  metallic  peroxides,  which  either  destroy  the  vitality  of 
bacteria  or  hinder  their  growth.  Silver,  zinc,  cadmium,  bismuth, 
and  copper,  have  this  action.  Consequently  silver  wire  and  foil, 
are  used  in  surgery  because  of  their  antiseptic  action.  Metallic 
fillings  in  teeth  prevent  the  growth  of  bacteria  that  cause  caries. 

Certain  cells  in  the  bodies  of  animals  (leucocytes)  and  some  ele- 
ments of  the  blood  serum,  being  bactericidal,  are  a  powerful  means 
of  internal  defense  against  infection. 

If  the  water  of  the  cytoplasm  of  bacterial  cells  is  dried  out,  the 
vitality  of  the  organism  suffers.  The  length  of  time  required  for 
drying  varies,  anthrax  spores  resisting  the  process  for  over  ten 


22  BACTERIA 

years.  Ancient  methods  of  preserving  foods  from  putrefying,  and 
which  are  still  in  vogue,  depend  upon  the  employment  of  some  of 
these  agents,  which  are  prejudicial  to  bacterial  life.  Meats  are 
salted,  pickled,  dried,  or  smoked.  Fruits  are  dried,  pickled,  or 
immersed  in  strong  saccharine  solution,  in  order  to  preserve  them 
from  decay,  in  every  instance,  the  absence  of  moisture,  the  excess 
of  salt,  sugar,  or  vinegar,  or  the  pyroligneous  acid  from  the  smok- 
ing, prevents  bacterial  growth,  and  consequently,  decay  of  the 
foodstuff.  The  products  of  bacterial  growths  often  inhibit,  or 
destroy,  the  cells  that  made  them,  as  well  as  other  bacteria. 
B.  pyocyaneus  and  S.  cholera,  have  this  property  of  secreting 
autolytic  ferments. 


CHAPTER  II 
PRODUCTS  OF  BACTERIAL  ENERGY 

According  to  their  chemical  activities,  bacteria  are  arbitrarily 
divided  into  the  following  classes: 

Photogens  Chromogens  Zymogens 

Saprogens  Aero  gens  Pathogens 

Photogens  are  those  bacteria  of  the  sea,  putrefying  flesh,  and 
damp  rotten  wood,  that  produce  a  faint  phosphorescence. 

Chromogens  are  bacteria  that  produce  colors  as  they  grow,  nota- 
ble among  which  may  be  mentioned  the  Staphylococcus  aureus, 
that  are  golden  in  hue;  B.  pyocyaneus,  of  a  greenish-blue;  and 
B.  prodigiosus  which  appears  a  brilliant  red. 

Zymogens  are  the  bacteria  of  fermentation,  which  is  the 
chemical  transformation  of  carbohydrates  by  the  action  of  bac- 
teria, with  the  evolution  of  CO2  CO  &  H.  Such  bacteria  are  use- 
ful in  the  industries  for  the  production  of  alcoholic  beverages, 
wine,  beer,  etc.  Through  the  actions  of  these  organisms  grape 
sugar  is  converted  into  alcohol,  lactic  acid,  and  acetic  acid. 

C6Hi2O6  =  2  C2H6O  +  2  CO2 

glucose      2  alcohol    2  carbonic  acid 
or 

C6Hi2O6  =  2  C3H6O6 

2  lactic  acid 
or 

C6H1206  =  3C2H402 

2  acetic  acid 
23 


24  PRODUCTS  OF  BACTERIAL  ENERGY 

From  the  bodies  of  ground  yeast  cells  a  soluble  ferment, 
Zymase,  has  been  expressed,  which  causes  alcoholic  fermentation 
of  cane  and  grape  sugars.  This  fact  proves  that  fermentation 
is  not  necessarily  a  vital  process.  The  fermentations  of  bacterial 
enzymes  may  give  acids,  and  also  aldehydes,  ketones,  CC>2, 
CO,  H,  N,  NH3,  marsh  gas  and  H2S.  The  carbohydrate  splitting 
powers  are  used  in  determinative  bacteriology. 

Fermentation  and  putrefaction  are  bacterial  enzymic  processes 
of  indispensible  importance  to  life.  Bacteria  reduce  excrementi- 
tious  matters  to  their  elements  and  then  others  build  up  these 
elements  into  conditions  favorable  for  plants.  This  process 
affects  the  cycle  of  utility  of  carbon,  sulphur  and  particularly 
nitrogen  in  the  air  and  soil.  Some  soil  bacteria  can  fix  nitrogen 
from  the  air  for  the  use  of  plants.  Because  of  the  importance  of 
these  processes,  culture  of  appropriate  bacteria  may  be  spread 
upon  exhausted  soil.  These  are  chiefly  nitrifying  bacteria. 
Manure  contains  the  denitrifying  organisms.  Bacterial  fermenta- 
tions produce  the  flavor  of  tobacco,  opium  and  butter. 

Enzyme  Production  by  Bacteria. — These  products  are  difficult 
to  define  because  few  have  been  obtained  in  an  entirely  pure 
state.  They  may  be  described  as  soluble,  but  non-dialyzable 
products,  precipitable  by  salts  of  heavy  metals  or  by  alcohol, 
destroyed  by  70°  but  resisting  drying  and  decomposition. 
They  are  restrained  by  excess  of  alkali,  of  acid,  and  by  an  accum- 
mulation  of  their  own  products.  Ferments  of  great  variety 
and  power  are  formed  by  the  zymogens,  as  proteolytic,  which  dis- 
solve proteids,  such  as  casein;  tryptic,  gelatine  liquefying;  diastase, 
which  converts  starch  into  sugar;  invertase,  which  changes  cane 
sugar  into  grape  sugar;  ferments  that  curde  the  casein  of  milk; 
and  it  may  well  be  that  the  activity  of  pathogenic  bacteria  in  the 
body  is  due  to  ferments  of  some  kind.  The  hemolytic  action  of 
the  golden  staphylococcus  or  the  tetanus  bacillus  is  thought,  by 
some,  to  be  of  enzymic  nature. 

Organized  ferments  (bacteria,  yeasts)  differ  from  the  unorganized 


SAPROGENS  AND  PATHOGENS  2$ 

(pepsin,  diastase).  The  latter  " exercise  solely  a  hydrolytic 
action"  (Fischer),  causing  the  molecules  of  insoluble  compounds 
to  take  up  water  and  to  separate  into  less  complex  molecules  of  a 
different  constitution,  which  are  soluble  in  water.  The  organized 
ones  act  differently.  Highly  complex  molecules  are  split  up,  and 
numerous  substances  of  a  totally  different  character  are  formed 
with  the  evolution  of  gases  and  by-products  (Fischer).  The 
reason  for  this  is,  perhaps,  to  be  found  in  the  supposition  that 
the  bacteria  abstract  oxygen  for  their  own  use,  and  thus  cause  the 
atoms  to  unite  into  an  entirely  different  substance.  According 
to  the  above-named  investigator,  it  is  not  possible  to  express 
such  chemical  changes  by  a  simple  equation.  Experiments  have 
shown  that  B.  typhosus  and  pyocyaneus  are  able  to  split  up  olive 
oil  or  fat,  and  produce  glycerine  and  fatty  acids,  thus  making 
them  accessible  to  fermentation  (Fischer).  The  action  of  the 
buttermilk  organisms,  while  usually  very  complex,  may  be 
represented  by  the  following : 

Ci2H22On  +  H2O  =  C6H12O6  +  C6Hi2O6 

lactose  galactose  dextrose 

C6Hi2O6  =  2C3H6O3 

galactose  lactic  acid 

Saprogens  produce  putrefaction  which  is  the  chemical  trans- 
formation of  albuminous  bodies  with  the  evolution  of  nitrogen, 
and  of  alkaloidal  substances,  known  as  ptomaines.  Aromatic 
elements  are  also  produced,  such  as  indol,  phenol,  kresol,  etc.  • 

It  is  therefore  obvious  that  fermentation  and  putrefaction  are 
separate  processes,  the  former  an  action  upon  carbohydrates, 
the  latter  a  splitting  up  of  proteins.  If  has  been  found  that  when 
organisms  can  attack  both  substances,  the  sugars  and  starches 
are  first  broken  up;  this  is  what  is  meant  when  it  is  stated  that 
carbohydrates  have  a  "sparing  action"  upon  proteins. 

Pathogens. — If  the  tissues  are  receptive  to  bacteria,  and  if  the 
latter,  in  any  way,  injure  the  tissues,  then  the  invading  organism 
is  called  pathogenic.  Theoretically  the  tissues  of  the  body  are 


26  PRODUCTS  OF  BACTERIAL  ENERGY 

sterile,  but  as  a  matter  of  fact,  isolated  pathogenic  bacteria 
such  as  colon  and  diphtheria  bacilli,  streptococci,  and  pneu- 
mococci,  have  been  found  in  the  tissues  and  cavities  of  the  body 
in  the  absence  of  pathological  evidence  of  their  presence. 

Sixteen  hours  after  death  the  blood  and  tissues  teem  with 
bacteria  that  have  wandered  in  from  the  intestines.  It  has  been 
shown  that  bacteria,  even  non-motile  ones,  can  migrate  through 
the  body  during  the  agonal  period. 

Bacteria  may  cause  disease  in  the  following  ways :  (a)  mechan- 
ically, a  clump  of  bacteria  may  plug  a  capillary;  (b)  simply  over- 
whelm the  tissues  and  absorb  the  oxygen  (anthrax);  (c)  they  may 
cause  new  growths  (tubercle) ;  or  false  membranes  to  form  in  the 
larynx  causing  suffocation  (diphtheria);  (d)  ulceration  of  heart 
valves  causing  cardiac  insufficiency;  (e)  thrombosis  in  the  veins 
and  arteries;  (J)  pus  formation;  (g)  by  generating  toxins  that 
cause  anaemias,  or  degeneration  of  important  elements  of  the 
nervous  system,  parenchymatous  organs  and  the  walls  of  the 
blood-vessels. 

The  tissues  of  certain  animals  are  receptive  for  particular 
bacteria,  and  the  latter  are  therefore  pathogenic  to  that  animal. 
B.  of  swine  plague  is  pathogenic  to  swine,  but  not  to  man.  B. 
typhosus  is  pathogenic  for  man,  but  not  to  swine. 

As  emphasized  above,  the  activities  of  bacteria  are  due  to  the 
enzymes  they  produce.  In  the  course  of  their  life,  bodies,  called 
toxins,  are  formed  that  have  the  power  of  producing  illness  in 
higher  plants  and  animals.  These  bodies  are  similar  to  the 
enzymes.  Both  are  produced  in  minute  quantities.  Their  exact 
chemistry  is  not  known,  and  pure  toxins,  at  least,  have  probably 
never  been  isolated.  We  test  for  them  by  animal  experiments 
while  the  presence  of  enzymes  may  be  observed  upon  artificial 
culture  media.  Toxins  of  bacteria  are  not  the  only  ones  formed. 
Castor  bean  produces  a  body  classed  among  the  toxins  as  does 
the  rattlesnake  in  its  venom.  These  bodies  differ  from  ptomaines, 
also  poisons,  by  being  less  resistant  to  heat,  in  causing  a  peculiar 


TOXINS  27 

blood  reaction  and  by  refusing  isolation.  The  toxins  are  not 
essential  to  the  life  of  pathogenic  bacteria  and  some  of  the  usually 
virulent  organisms  may  grow  without  toxin  development.  Toxin 
productions  may  be  lost  and  regained.  The  real  object  of  the 
toxins  is  not  known,  as  it  is  not  thought  that  bacteria  gain  any- 
thing by  producing  disease.  They  are  separate  from  the  other 
chemical  bacterial  products.  Toxins  may  be  divided  into  those 
which  are  secreted  through  the  bacterial  cell  wall  and  diffuse 
through  the  median  in  which  organisms  are  growing,  the  extra- 
cellular or  soluble  toxins,  and  those  which  remain  within  the 
bacterial  cells  and  are  only  liberated  upon  their  death  and  dis- 
integration, the  endotoxins.  Closely  related  to  the  second  class 
are  the  so-called  toxic  bacterial  proteins  or  plasmins.  These  do 
not  separate  from  the  structures  since  bacteria  which  produce 
them  furnish  a  toxic  mass  if  thoroughly  washed,  ground  and 
rewashed. 

Examples  and  Characters.    Soluble  or  Extracellular  Toxins. — 

The  best  examples  are  those  of  the  tetanus  and  diphtheria  bacilli. 

In  diseases  caused  by  these  germs,  bacteria  do  not  enter  the  body 

i  fluids  but  the  general  manifestations  are  due  to  absorbed  soluble 

I  poisons.     Such  toxins  are  soluble  in  water;  they  are  rendered  inert 

i  by  heating,  sunlight  and  some  chemicals.     They  dialyze  very 

i  slowly  and  are  not  crystallizable.     They  may  be  precipitated  with 

j  the  albumen  fraction  of  the  medium.     They  may  be  precipitated 

and  dried,  in  which  state  they  keep  much  longer  than  when  in 

solution,  and  then  are  more  resistant  to  heat.     Curiously  enough 

I  the  toxins  may  be  destroyed  by  proteoly  tic  enzymes.     Some  toxins 

!  are  complex;  the  tetanus  toxin  for  example,  contains  two  elements, 

;  one  a  dissolving  power  on  red  blood  cells,  the  other  a  stimulator 

i  of  the  motor  system.     They  are  specific  for  each  organism. 

Endotoxins. — These  are  exemplified  by  the  poisons  of  the  ty- 
phoid and  plague  organisms.  We  know  little  of  their  chemistry 
but  we  may  assume  that  it  is  of  protein  material  and  similar  to 
that  of  the  bacterial  cell.  These  toxins  are  less  rigidly  specific 


28  PRODUCTS   OF  BACTERIAL   ENERGY 

than  the  extracellular  poisons.  They  are  probably  quite  complex 
in  activity  as  they  give  rise  to  various  anti-poisons  when  in  the 
animal  body.  These  poisons  are  resistant  to  heating  at  8o°C.  and 
keep  under  artifical  conditions  much  longer  than  soluble  toxins. 

The  toxic  bacterial  proteins  are  best  exemplified  by  tuberculin. 
This  is  complex  mixture  of  the  proximal  principles  of  the  tubercle 
bacillus  and  is  probably  albuminose  in  character.  These  sub- 
stances are  almost  as  specific  for  their  own  germs  as  the  toxins  and 
much  more  so  than  the  endotoxins.  They  are  capable  of  produc- 
ing a  reaction  in  animals  similar  to  that  which  might  be  produced 
by  the  organisms  themselves.  For  example  tuberculin,  wholly 
free  from  tubercle  bacilli,  will  produce  a  reddening  of  the  skin  or  a 
rise  of  temperature  if  injected  into  a  tuberculous  individual.  The 
dead  tubercle  bacillary  mass  if  placed  beneath  the  skin  of  a  healthy 
guinea  pig  will  set  up  a  local  limited  miliary  tubercle.  The 
reactions  from  mallein  and  luetin  (q.v.)  injection  are  due  to  toxic 
proteins.  The  proteins  are  usually  thermostable,  that  is  not 
destroyed  at  ioo°C.;  this  is  also  called  coctostabile. 

In  practice  it  may  not  be  so  simple  to  separate  bacteria  that 
produce  the  various  poisonous  elements  as  the  above  descriptions 
would  indicate.  Toxins  are  all  in  a  sense  specific,  that  is  they 
are  for  the  most  part  selective  in  action,  and  are  harmless  if 
swallowed.  The  diphtheria  toxin  is  absorbed  from  a  raw  inflamed 
surface  under  cover  of  an  exudate  composed  of  fibrin  and  bacteria. 
The  tetanus  toxin  is  absorbed  from  its  seal  of  manufacture  in  the 
depths  of  a  punctured  wound.  The  endotoxin  of  typhoid  bacilli 
has  no  pathogenic  effect  if  swallowed  or  rubbed  in  skin  or  mucous 
membrane.  If  it  be  injected  under  the  skin  in  the  absence  of 
bacteria  it  will  call  forth  reactions  on  the  part  of  the  body  similar 
to  those  expressed  when  living  typhoid  germs  are  circulating. 
Toxins  are  again  relative  in  their  affinities.  Tetanus  toxin  is  fatal 
for  man  and  horses  while  rats  and  birds  are  resistant  to  it.  We 
use  this  expression  of  specificity  for  determining  the  nature  of 
certain  germs.  We  may  speak  of  failures  to  react  as  failures 


TOXINS  29 

of  receptivity  on  the  part  both  of  the  microbe  and  the  injected 
animal. 

Other  characters  of  toxins  are  that  they  act  in  dilute  suspen- 
sions, are  destroyed  by  heat,  and  produce,  when  injected  in  small 
doses  into  animals,  a  specific  anti-substance. 


CHAPTER  III 
INFECTION 

Infection  means  the  successful  invasion  of  the  tissues  of  the 
body  by  either  animal  (protozoa,  vermes)  or  vegetable  (bacteria 
and  moulds)  organisms  with  the  evidences  of  their  action.  To 
successfully  infect  the  body,  bacteria  must  enter  the  tissues,  be 
of  sufficient  number,  find  the  tissues  receptive,  and  continue 
to  multiply. 

The  skin,  mucous  membranes,  and  the  various  cavities  of  the 
body  connected  with  the  outside  air,  teem  with  countless  bacteria 
at  all  times,  many  of  which  are  pathogenic,  yet  there  is  no  infec- 
tion, because  the  tissues  are  not  invaded.  Again,  there  can  be  no 
doubt  that  highly  pathogenic  bacteria  enter  the  tissues  of  healthy 
people  at  times,  in  small  numbers,  and  yet  no  disease  is  produced, 
because  of  their  scarcity,  or  by  reason  of  the  tissues  not  being 
receptive.  Infection  implies  not  only  invasion  of  the  body,  but 
injury  to  the  tissue.  Certain  bacteria  may  invade  a  body,  and 
yet  create  no  harm.  These  bacteria  may  enter  dead  or  dying 
body  tissues,  and  secrete  poisonous  substances  (toxins)  which 
may  be  absorbed,  and  produce  pathologic  symptoms  known  as 
Saprcemia.  Clots  of  blood  in  the  parturient  uterus,  and  gan- 
grenous limbs  may  be  invaded  by  strict  saprophytes  incapable  of 
life  in  living  tissues,  and  yet  cause  much  harm  by  the  absorption 
of  their  products. 

Infestation  is  when  organisms,  even  pathogenic,  are  present  in 
a  place  without  exciting  a  reaction;  the  term  is  best  used  however 
to  imply  the  presence  and  action  of  animal  parasites.  Matter 
carrying  pathogenic  germs  is  called  infective. 

30 


Depending  upon  the  ability  to  grow  in  the  body,  bacteria  may 
be  divided  into:  (i)  purely  saprophytic  ;  (2)  occasionally  para- 
sitic; <ind  (3)  purely  parasitic.  A  host  harbors  a  parasite. 

Purely  saprophytic  germs  cannot  live  in  tissues  at  all;  those 
that  are  occasionally  parasitic  lead  a  saprophytic  existence  in 
the  soil  or  water,  and  yet  may  invade  the  body,  and  produce 
disease:  the  tetanus  and  malignant  oedema  bacilli  are  examples 
of  this  group.  Those  bacteria  that  are  purely  parasitic  are  only 
known  as  they  exist  in  the  tissues  of  the  infected  host,  and  have 
no  outside  existence  at  all. 

Koch's  Postulates 

In  order  to  prove  that  a  certain  organism  is  the  infectious  agent 
of  a  given  disease,  Koch  has  devised  four  postulates  which  the 
given  organism  must  fulfill  before  it  can  be  considered  the  cause 
of  the  disease. 

1.  The  organism  must  be  found  microscopically  in  the  tissues 
of  the  animal  having  the  disease,  and  its  position  in  the  lesion 
should  explain  the  latter. 

2.  It  must  be  isolated  in  pure  state  from  bodies  of  the  diseased 
animals. 

3.  And  then  it  must  be  grown  for  successive  generations  in 
culture  media. 

4.  If  injected  into  a  healthy  animal,  or  animals,  it  must  produce 
the  same  disease,  and  be  found  in  the  lesions  of  the  disease  in 
the  animal's  tissues. 

Some  of  the  many  organisms  that  certainly  fulfill  these  condi- 
tions, are  as  follows : 

Streptococcus  Pyogenes  (Sepsis).  Actinomyces. 

B.  of  Tuberculosis.  B.  of  Diphtheria. 

B.  of  Anthrax.  B.  of  Tetanus. 

B.  of  Glanders.  B.  of  Malignant  (Edema 

B.  of  Bubonic  Plague.  B.  of  Malta  Fever. 


32  INFECTION 

B.  of  Typhoid.  B.  of  Dysentery. 

Spirillum  Choleras.  Meningococcus. 

Pneumococcus  (Pneumonia) . 

Spiroch&ta  of  Relapsing  Fever  and  of  Syphilis 

There  are  several  other  organisms  that  are  considered  to  be 
the  cause  of  specific  disease,  but  they  do  not  fulfill  the  postulates. 
Among  these  are : 

The  Protozoa  of  Malarial  Fever 
Amoeba  Dysenteries. 

While  the  specifications  outlined  by  Koch  as  indicating  the 
etiological  role  of  an  organism  were  sufficient  for  the  period  at 
which  they  were  laid  down,  advances  in  immunology  have  added 
so  much  information  about  antigens  and  antibodies  that  it  is 
but  right  today  to  expect  that  a  virus  should  behave  as  an 
antigen  by  calling  forth  certain  immunity  reaction  under  spon- 
taneous and  experimental  conditions.  Such  as  expectation  is 
fulfilled  in  practically  all  cases,  and  indeed  has  been,  even  in 
a  few  instances  where  all  Koch's  postulates  could  not  be  com- 
pleted, typhoid  fever  being  a  notable  example. 

In  rheumatic  fever,  measles,  mumps,  yellow  fever,  chicken- 
pox,  rabies,  and  dengue,  the  specific  cause  has,  thus  far,  eluded 
discovery.  In  the  case  of  measles,  hog  cholera,  and  some  of 
the  eruptive  diseases,  it  has  been  found  that  the  cause  of  these 
diseases  resides  in  the  blood,  and  if  the  serum  of  the  latter  is 
carefully  filtered  through  a  Berkefeld  filter,  it  is  still  capable  of 
producing  the  disease  in  susceptible  animals.  Careful  micro- 
scopic search  fails  to  show  any  bodies  in  the  serum  that  might  be 
considered  the  agents  of  infection,  and  it  is  thought  that  these 
organisms  are  submicroscopic  (see  chapter  on  Filterable  Viruses). 

If  the  invading  organism  is  a  pure  saprophyte  the  various" 
forces  for  internal  defense  immediately  act  upon  and  destroy  it. 

Bacteria  are  disposed  of  in  diverse  ways.     By  means  of  the 


ATTENUATION   OF  BACTERIA  33 

lymph  channels  they  are  carried  to  the  various  mucous  surfaces 
of  the  body,  intestinal  and  bronchial.  The  liver,  according  to 
Adami,  destroys  at  once  bacteria  absorbed  from  the  intestines. 
During  typhoid  fever,  the  typhoid  bacilli  are  often  found  in 
the  urine,  the  organisms  escaping  from  the  blood  or  from  the 
lymphoid  foci  in  the  kidney.  Pathogenic  bacteria  are  discharged 
from  the  body  in  feces,  pus,  sputum,  and  in  scales  in  the  des- 
quamating skin  diseases. 

To  successfully  inoculate  a  guinea  pig  with  tuberculosis,  the 
tubercle  bacilli  should  be  injected  beneath  the  skin. 

It  has  been  said  that  successful  invasion  demands  a  sufficient 
number  of  organism;  it  is  equally  true  that  the  number  admitted 
will  determine  the  character  of  disease  to  arise,  as  is  indicated 
by  the  following  observation  of  Cheyne. 

In  experimenting  with  the  staphylococcus  aureus,  it  was  found 
that  250,000,000  were  required  to  cause  an  abscess;  and  1,000,000,- 
ooo  were  needed  to  cause  death.  The  internal  powers  of  defense 
were  able  to  cope  with  or  limit  the  action  of  a  few  million  to  a 
certain  locality,  but  could  not  withstand  the  injection  of  over- 
whelming numbers,  which  caused  the  animal's  death. 

There  are  three  attributes  which  make  successful  the  invasion 
of  pathogenic  germs  into  the  body:  virulence,  toxicity  and 
pathogenicity.  These  factors  shade  into  each  other  sometimes 
very  confu singly  and  are  of  course  capable  of  varying  proportions 
in  the  same  organism.  In  order  to  combat  successfully  the  pri- 
mary defenses  of  the  body,  a  virus  requires  virulence,  the  degree 
and  permanency  with  this  is  accomplished  being  due  to  the  amount 
of  poison  the  invader  can  elaborate  to  keep  the  safety  devices 
of  the  economy  from  conquering.  The  physical  damage  done 
is  attributable  to  the  pathogenicity  of  the  germ.  It  is  like  a 
fight  where  the  man  has  strength,  and  staying  powers  and  does 
physical  damage  to  his  adversary. 

Ehrlich's  explanation  of  virulence  assumes  that  bacteria  have 
binding  posts  or  receptors  and  the  more  of  these  a  germ  has,  the 


34  INFECTION 

more  of  the  natural  defenses  it  can  anchor  and  remove  from  the 
field. 

Their  virulence  can  be  lessened  by  cultivation  at  a  higher 
temperature  than  the  body,  42.5°-47°C.;  by  drying;  the  exposure 
to  light;  the  action  of  chemicals;  compressed  oxygen;  and  by 
passing  the  organism  through  the  bodies  of  non-susceptible 
animals.  The  attenuation  or  weakening  of  the  pathogenic  powers 
of  bacteria  is  useful  for  the  production  of  various  vaccines  which 
are  valuable  in  preventive  medicine. 

By  growing  the  anthrax  bacillus  atahigh  temperature,  42.5°C., 
it  becomes  so  avirulent  that  it  is  incapable  of  destroying  sheep  or 
rabbits.  It  is  then  used  as  a  vaccine  to  prevent  infection  with 
virulent  bacilli.  By  exposing  the  spinal  cords  of  animals  dead 
from  hydrophobia  to  the  action  of  drying  for  various  periods, 
Pasteur  was  able  to  attenuate  the  virus,  so  that  it  would  not 
produce  hydrophobia,  but  on  the  contrary,  it,  by  repeated 
inoculation,  caused  immunity.  The  inoculation  of  monkeys 
(which  are  non-susceptible)  with  hydrophobia  virus  attenuates 
it.  The  growth  of  the  small-pox  organism  in  the  cow,  causing 
cow-pox,  so  reduces  the  virulence  of  the  germ  that  it  is  incapable 
of  producing  small-pox  in  man,  but  only  vaccinia;  infection  with 
this  gives  immunity  against  small-pox.  The  flesh  of  animals 
that  have  died  from  quarter-evil  is  so  changed  by  heat  and  desic  - 
cation  that  if  it  is  injected  into  susceptible  animals,  they  do  not 
succumb  but  are  vaccinated  against  infection  with  the  virulent 
organism. 

When  we  speak  of  attenuation  of  virulence  we  usually  refer  to 
the  effects  on  experimental  animals  and  specify  what  attenuation 
is  meant  when  they  are  to  be  used  as  vaccine.  A  very  interesting 
pathogenic,  yet  attenuated,  form  of  streptococcus  is  to  be  met 
in  subacute  endocarditis.  These  organisms  produce  serious  or 
even  fatal  valvulitis,  and  yet  have  no  effect  upon  lower  animals. 
They  are  extremely  hard  to  remove  from  the  body.  They 
have  accustomed  themselves  to  residence  in  the  body,  have  estab- 


AVENUES   OF   INFECTION  35 

lished  a  balance  of  poise  between  their  offenses  and  the  bodily 
defenses  and  practically  cannot  be  rapidly  dislodged.  These  are 
called  fixed  or  fast  strains.  Such  strains  may  be  seen  under  other 
conditions  such  as  the  typhoid  bacillus  in  the  gall-bladder.  These 
fast  strains  usually  are  found  at  places  remote  from  intimate  con- 
tact with  the  defenses  of  the  body,  the  leucocytes  and  blood  serum 
as  in  the  cases  cited. 

The  malignancy  of  bacteria  may  be  heightened  in  various  ways : 
(i)  By  passing  them  repeatedly  through  the  bodies  of  susceptible 
animals;  (2)  by  cultivation  in  culture  media  in  collodion  sacs 
placed  in  the  abdominal  cavities  of  animals;  (3)  by  injections 
mixed  with  other  injurious  substances,  such  as  lactic  acid,  and  the 
metabolic  products  of  foreign  bacteria.  Cultures  of  pneumococci 
may  be  made  so  virulent  by  the  first  means  that  only  one  pneu- 
mococcus  is  capable  of  setting  up  a  fatal  septicaemia  in  a  rabbit. 
By  injecting  attenuated  diphtheria  bacilli  with  streptococci  into  a 
rabbit,  the  virulence  of  the  bacilli  can  be  raised,  as  mixed  infection 
often  adds  to  the  virulence  of  an  organism.  Malignant  strepto- 
coccic  infection  added  to  virulent  diphtheria  infection,  greatly 
increases  the  severity  of  the  disease.  The  transference  of  infec- 
tive agents  from  one  person  to  another  during  an  epidemic 
increases  the  virulent  action  of  the  organism  by  reason  of  the 
rapid  passage  from  individual  to  individual. 

Mixed  infections  are  those  in  which  more  than  one  kind  of 
virus  is  active.  It  is  of  course  possible  that  two  kinds  may 
originate  a  disease,  but  it  is  usual  for  one  germ  to  initiate  a  process 
and  another  to  be  superimposed  upon  it,  usually  intensifying  the 
lesions.  The  active  ulcerative  inflammation  in  tuberculous  lungs 
is  usually  due  to  be  secondary  effect  of  streptococci. 

The  secondary  streptococcic  infection  in  small-pox  and  in 
phthisis  complicates  the  primary  infection  and  frequently  causes 
death  of  the  individual  affected. 

The  avenue  of  infection  and  the  tissues  infected  alter  the  type  of 
the  disease  exceedingly.  Streptococci  invading  the  tonsils  cause 


36  INFECTION 

tonsillitis,  but  the  same  organisms  entering  the  skin  cause  erysipe- 
las of  phlegmons;  or  if  the  uterus  is  infected  after  the  birth  of  a 
child  the  disease  is  still  different  and  more  serious.  If  the  tubercle 
bacilli  enter  the  skin  they  produce  lupus;  if  swallowed  they  cause 
ulceration  of  the  bowels,  and  subsequently  invade  the  peritoneum; 
if  inhaled,  tuberculosis  of  the  air  passages,  phthisis,  or  tuberculous 
laryngitis  may  follow.  If  cholera  spirilla  be  injected  into  a  vein 
of  a  guinea  pig,  it  may  develop  choleraic  septicaemia;  if  they  are 
injected  into  the  peritoneal  cavity,  a  choleraic  inflammation  of  the 
peritoneum  is  produced,  and  not  a  septicaemia.  Pneumococci  if 
injected  into  a  vein  cause  a  rapid  septicaemia,  or  they  may  give 
rise  to  abscesses  anywhere  in  the  body.  Like  streptococci,  they 
may  be  the  cause  of  inflammation  in  any  tissue,  particularly 
serous  membranes,  and  show  different  clinical  entities,  according 
to  the  organs  involved,  and  the  morbid  anatomy  and  physiology 
produced.  The  fatality  of  a  bacterial  infection  varies  with  the 
avenue  of  inoculation:  it  is  safer  to  have  a  skin  infection  than  a 
meningeal,  or  endocardial  one,  not  only  from  the  likelihood  of 
rapid  toxin  absorption,  but  from  purely  mechanical  damage,  as 
pressure  and  interference  with  vital  functions  by  inflammatory 
products  such  as  fibrin,  tubercles,  serum  and  pus. 

How  Bacteria  Are  Brought  to  the  Body. — Air-borne  infection 
may  occur  by  the  direct  transference  of  the  bare  organisms,  a  very 
rare  occurrence,  or  by  dust  or  by  droplets  of  fluid  usually  sputum. 
Organisms  settle  on  objects  of  our  environment  when  leaving  the 
sick  and  can  be  stirred  up  with  the  dust.  This  is  important  for 
diphtheria,  the  acute  exanthemata  and  tuberculosis  although 
the 'most  dangerous  source  for  the  last  is  the  coughing  con- 
sumptive. The  transmission  of  pertussis  and  pneumonia  is  almost 
surely  always  a  droplet  convection. 

Water-borne  infection,  including  typhoid,  cholera  and  dysen- 
tery occurs  by  the  contamination  of  water  courses  with  the  dis- 
charges of  the  respective  diseases. 

Milk-borne   diseases,   tuberculosis,  diphtheria,  epidemic  sore 


SOURCES   OF  INFECTION  37 

throat  and  some  others,  occur  because  persons  or  animals  suffering 
with  the  disease  have  handled  or  supplied  the  milk.  This  direct 
contamination  also  applies  to  food  like  meat  and  oysters  (typhoid 
and  meat  poisoning). 

Soil-borne  diseases  are  chiefly  those  arising  by  direct  implanta- 
tion of  earth  into  the  body.  Of  course  the  ground  may  be  soiled 
by  discharges  from  infectious  diseases  and  contamination  of 
hands  and  clothing. 

Animal  carriers  of  disease  include  those  acting  as  intermediate 
hosts  (anopheles  mosquito  in  malaria) ;  as  mechanical  conveyances 
of  a  direct  or  indirect  nature,  in  the  former  case  like  transmission 
of  organisms  from  a  sick  man  or  animal  to  a  well  one,  in  the  latter 
case  transferring  the  germs  to  food  consumed  by  a  healthy  being; 
or  acting  as  a  passive  host  for  the  germ  as  is  the  case  in  the 
transmission  of  plague  bacilli  by  the  rat  flea. 

Human  transmission  of  infective  matter  is  the  most  important 
of  all  methods  as  it  is  an  axiom  that  a  person  suffering  with  a 
disease  is  most  capable  of  transmitting  it.  This  occurs  by  direct 
contact,  by  the  carrier  state  and  by  passing  the  contagium  to  the 
embryo. 

Carriers. — After  recovery  from  certain  diseases,  notably  ty- 
phoid fever,  diphtheria  and  cholera,  convalescents  may  carry 
in  themselves  fully  virulent  germs  with  no  outward  evidences 
thereof.  Such  persons  are  called  " carriers"  and  are  of  the  highest 
importance  in  hygiene.  The  reasons  for  this  condition  are  several. 
These  germs  may  be  removed  from  the  bodily  defenses  or  the 
body  may  be  immune  to  them;  again  they  may  be  fixed  or  fast 
strains.  Wherever  they  are  they  may  escape  and  infect  another 
person.  After  typhoid  fever  bacilli  remain  in  the  gall-passages 
and  bladder;  after  cholera  in  the  deep  mucous  membranes  and 
after  diphtheria  the  crypts  of  the  tonsils  or  the  nasopharynx  may 
hold  them.  Vaccination  or  operation  may  be  needed  to  remove 
them.  Persons  never  known  to  have  had  enteric  fever  have 
been  known  to  harbor  bacilli  in  their  gall-bladder.  One  typhoid 


38  INFECTION 

carrier,  " Typhoid  Mary"  a  cook,  is  known  to  have  infected  26 
persons.  Such  persons  because  of  their  apparent  innocence 
might  be  called  "  hidden  carriers."  They  have  been  found  trans- 
mitting dysentery  and  poliomyelitis  as  well  as  the  above  typhoid 
fever,  and,  judging  from  the  continued  existence  of  the  exanthe- 
mata in  cities,  it  may  be  that  we  shall  find  such  persons  harboring 
the  virus  of  varicella,  mumps  and  pertussis. 

Local  Immunity  to  Infection. — There  is  evidently  more  resistance 
offered  by  the  liver  against  invasion  than  by  the  peritoneum.  It 
is  not  likely  that  a  man  would  contract  typhoid  through  skin 
infection,  nor  is  it  probable  that  he  would  contract  tetanus  by 
swallowing  tetanus  bacilli,  but  the  reverse  of  these  conditions 
certainly  produces  infection. 

Infection  may  be  caused  from  without  the  body,  or  from  within. 
Lockjaw,  sepsis,  hydrophobia,  or  anthrax  may  follow  injuries  from 
rusty  nails,  splinters,  weapons,  unsterile  fingers,  or  instruments. 
Personal  intercourse,  bites,  kisses,  sexual  intercourse,  association 
with  persons  suffering  from  exanthematous  or  contagious  diseases 
may  transmit  disease. 

Winslow  has  found  colon  bacilli  upon  9  percent  of  the  hands  he 
examined.  Tubercle  bacilli  have  been  found  on  the  hands  of  the 
non-tuberculous.  Some  organisms,  notably  the  smegma  bacillus, 
pyocyaneus  bacilli  and  cocci  resembling  the  white  pus  former,  may 
be  said  to  be  normal  inhabitants  of  the  skin. 

The  bites  of  insects  that  are  intermediate  hosts  of  infectious 
agents  (plague  bacilli,  malarial  organisms,  etc.)  are  sources  of 
infection  from  without,  as  is  also  the  ingestion  of  infected  food  or 
water. 

Infection  from  within  may  be  caused  by  the  migration  of  bac- 
teria from  the  skin  inwards,  or  from  any  of  the  mucous  membranes, 
on  which,  and  in  which  many  pathogenic  bacteria  at  all  times  may 
be  found. 

Bacteria  from  the  mouth,  stomach  intestines  and  the  rectum 
may  invade  the  tissues  and  the  blood  under  certain  conditions. 


SOURCES   OF   INFECTION  39 

This  is  particularly  the  case  during  the  last  stages  of  diseases,  not 
necessarily  infectious,  such  as  chronic  heart  disease,  kidney 
disease,  or  diabetes.  Vital  resistance  is  much  lowered,  and  intes- 
tinal bacteria,  invading  the  tissues  in  enormous  numbers,  set  up 
what  is  known  as  terminal  infection,  which  is  often  the  immediate 
cause  of  death. 

The  stomach  with  its  gastric  juice,  containing  during  digestion 
.2  percent  to  .3  percent  of  hydrochloric  acid,  guards  the  lower  ali- 
mentary tract  against  infection.  A  great  many  bacteria  are  in- 
gested with  foods,  particularly  with  milk,  cheese,  and  overripe 
fruit.  These  in  the  most  part  are  quickly  destroyed  by  the  hy- 
drochloric acid.  When  the  stomach  is  diseased  and  the  contents 
become  stagnant,  as  in  stenosis  of  the  pylorus,  and  in  carcinoma, 
when  HC1  is  diminished,  or  absent,  fermentative  bacteria  give  rise 
to  great  amount  of  gas,  and  lactic  acid,  to  the  great  discomfort  of 
the  patient.  The  normal  acidity  of  the  stomach  is  a  great 
safeguard  against  infection  with  cholera.  If  tubercle  bacilli  are 
swallowed,  and  if  infection  occurs,  the  lesion  is  not  always  localized 
to  the  alimentary  tract.  Lesions  of  the  lymph  glands,  peritoneum, 
bones,  and  nervous  tissues  often  follow  the  ingestion  of  these  or- 
ganisms. Dogs  fed  on  soup  containing  great  numbers  of  tubercle 
bacilli,  and  then  killed  three  hours  after,  were  found  to  have  bacilli 
in  the  thoracic  duct.  Chyle  from  the  duct,  injected  into  guinea 
pigs,  caused  tuberculosis  in  them  (Nicolas  and  Descos). 

The  interior  of  the  uterus,  the  bladder,  urine,  deep  urethra,  and 
lungs  are  generally  sterile  in  health.  With  the  exceptions  noted 
where  germs  are  not  usually  found,  all  tissues,  especially  the  inlets 
and  outlets  of  the  body,  may  be  said  to  have  a  normal  bacterial 
flora.  Bile  has  a  distinct  antibacterial  power,  which  indeed  is  one 
of  its  functions  in  the  intestines. 

The  placenta  is  an  avenue  of  infection  in  several  diseases:  not- 
ably small-pox,  anthrax,  glanders,  typhoid  fever,  and  sometimes 
tuberculosis  pass  through  the  placenta  from  mother  to  foetus. 
Streptococci  may  pass  through  the  placenta  of  a  woman  with 


40  INFECTION 

ante-delivery  sepsis  and  cause  peritonitis  in  the  child.  Recurrent 
fever  has  been  transmitted  from  mother  to  foetus,  and  the  specific 
spirillum  has  been  detected  in  the  latter's  blood. 

A  case  has  been  recorded  in  which  a  woman  suffering  from 
pneumonia  gave  birth  to  a  child,  which  died  thirty-six  hours  after- 
ward, and  autopsy  revealed  a  consolidation  of  the  lower  left  lung, 
and  microscopic  examination  discovered  pneumococci.  A  hydro- 
phobic  cow  was  delivered  of  a  calf  that  developed  rabies  three  days 
after  birth. 

McFarland  divides  microbic  infection  in  three  heads : 

Phlogistic. — Characterized  by  restricted  growth  and  local 
irritation. 

Toxic. — Characterized  by  restricted  growth  and  toxin  dissemi- 
nation. 

Septic. — Characterized  by  unrestricted  growth  in  the  blood  and 
lymph.  In  the  three  groups,  the  damage  is  done  ultimately,  by 
metabolic  products  acting  on  the  tissues.  If  the  product  be  not 
soluble  the  harm  done  is  purely  local,  as  in  the  formation  of  tuber- 
cles by  the  toxin  of  the  tubercle  bacilli. 

If  the  growth  be  restricted,  as  in  tetanus  and  diphtheria,  the 
toxin  being  soluble  and  diffusible,  harm  is  done  to  tissues  remote 
from  the  infected  area. 

Anthrax  and  streptococci  and  other  pus  organisms  by  rapid 
increase  in  the  blood  eventually  infect  all  the  tissues. 

Combinations  of  these  forms  of  infection  may  be  at  first  confined 
to  some  particular  area;  the  pneumococcus,  which  is  generally 
restricted  to  the  lungs  at  the  outset,  may  ultimately  infect  the 
blood,  causing  septicaemia  and  localized  lesions  in  more  or  less 
remote  parts,  such  as  the  veins  of  the  leg,  or  inflammation  of  the 
meninges.  In  a  topographical  sense  infection  may  be  local,  focal 
and  general.  Local  disease  is  limited  in  extent  and  at  most  gives 
only  trifling  general  manifestations  by  absorption  of  products  of 
inflammation,  a  boil.  When  an  infection  becomes  well  established 
in  a  small  locality  but  without  active  general  evidences,  it  may 


SOURCES   OF   INFECTION  41 

still  send  out  a  few  organisms  or  small  quantities  of  poison  which 
can  attack  other  parts.  Thus  from  a  root  abscess,  germs  may 
sneak  into  the  blood  stream  and  settle  in  the  kidneys  or  joint 
membranes;  this  is  focal  infection  and  is  usually  subacute  or 
chronic  in  character.  General  inffection  is  self  explanatory. 

Bacteria  may  become  accustomed  to  the  fluids  of  the  body  by  a 
similar  process  and  may  elaborate  free  receptors  or  their  own 
protection,  i.e.,  anti-bacteriolysins  (Welch's  theory). 

In  the  aged,  and  in  chronic  disease  of  the  liver  and  kidneys, 
the  complement  existing  in  the  blood  may  become  reduced  in 
quantity,  and  the  individual  may  succumb  to  an  infection,  which 
ordinarily  would  be  mild. 

Soluble  products  of  bacterial  activity  which  are  alkaloidal 
(basic),  crystalline  in  character,  and  mostly  poisonous,  are  known 
as  ptomaines,  or  putrefaction  alkaloids.  They  are  highly  com- 
plex in  chemical  structure,  and  are  difficult  to  isolate. 

The  foregoing  processes  are  due  to  the  toxic  products  of  bac- 
teria during  their  growth  in  the  tissues,  substances  mentioned 
before  but  now  deserving  a  more  detailed  study  from  the  stand- 
point of  the  diseased  tissue. 

Bacterial  endotoxins  are  poisonous  substances  liberated  only 
upon  the  death  and  disintegration  of  the  germ  cells.  They  are 
moderately  active  in  attacking  wandering  and  special  tissue 
cells;  they  are  more  resistant  to  heat  and  ferments  than  toxins. 
The  identity  of  these  toxins  has  given  rise  to  considerable  dispute 
and  there  will  be  given  the  two  principle  ideas  concerning  them. 
The  older  theory  considered  them  integral  parts  of  the  cell,  peculiar 
to  each  virus  and  calling  forth  specific  responses  because  of  the 
individuality  of  each  toxin.  Recent  work  has  shown  that  the 
chemical  and  immunological  response  to  bacterial  injection  is 
similar  to  that  obtained  by  the  use  of  serum,  or  egg  white  or 
animal  cells.  For  this  and  more  minute  reasons  it  is  believed  by 
some  that  there  is  in  every  protein  (bacterium,  cell,  serum)  a 
toxic  part  with  a  common  construction.  Another  and  peculiar 


42  INFECTION 

moiety  for  each  is  non-toxic  but  represents  the  part  which  calls 
forth  specific  antibodies.  The  characteristic  infectious  phenomena 
of  each  disease  are  therefore  due  to  the  non-toxic  part  of  its 
molecular  construction.  It  has  been  shown  that  protein 
cleavage  products  can  cause  fever  if  injected  into  animals. 

Cholera  and  typhoid  organisms  do  not  produce  soluble  toxins 
in  the  body,  but  when  they  are  disintegrated  therein,  soluble 
poisons  (intracellular)  are  liberated. 

Bacterioprotein  or  plasmins  are  albuminous  bodies  produced 
by  bacteria,  are  not  altered  by  heat,  and  produce  fever  and  in- 
flammation. The  best  examples  of  these  are  mallein  a  product 
obtained  from  old  cultures  of  glanders  bacilli,  and  the  original, 
or  old  tuberculin  of  Koch. 

Toxins  or  toxalbumins  are  soluble,  non-crsytallizable,  non- 
dialyzable  bacterial  products  which  are  removable  by  filtration 
from  the  bacteria,  and  which  are  thermolabile. 

These  various  poisons  produce  many  of  the  clinical  pathological 
entities  and  symptoms,  known  to  physicians.  Their  highly 
complex  molecular  structure  enables  a  group  of  atoms  in  the 
toxic  molecule  to  unite  with  a  certain  other  group  of  atoms  in 
the  protoplasmic  molecule  of  a  body  cell.  The  latter  is  either 
killed  outright,  or  else  is  stimulated  to  produce  other  free  groups 
of  combining  atoms  (lateral  chains)  which  may  unite  with  other 
toxic  groups. 

Various  kinds  of  cells  are  attacked  in  infective  processes. 
Leucocytes  may  be  degenerated,  forming  pus;  red  blood  cells  may 
be  dissolved,  causing  anaemia;  important  nerves  may  be  degener- 
ated; or  muscle  fibers  of  the  heart  may  undergo  fatty  degeneration 
and  die.  Again,  mechanically  important  serous  cavities  may  be 
filled  with  serum,  interfering  with  normal  functions  of  the  en- 
veloped organs.  The  heart  orifices  may  be  closed  partially  or 
emboli  may  form,  or  false  membranes  block  the  air  passages,  and 
a  hundred  other  pathological  changes  may  be  wrought  by  these 
toxins. 


TOXINS    OR    TOXALBUMINS  43 

Most  toxins  are  easily  decomposed  by  sunlight,  air,  and  heat. 
Absolute  alcohol  separates  the  active  principles  from  the  bouillon 
in  which  it  grows.  Ammonium  sulphate  also  precipitates  the 
toxins  from  cultures  of  tetanus  and  diphtheria  bacilli,  from  which 
they  may  be  collected,  dried  and  powdered,  and  in  this  state 
may  be  kept  much  longer  without  deteriorating  into  inert  sub- 
stances. Small  quantities  of  bile  and  pancreatic  juice  destroy 
the  toxic  properties  of  diphtheria  and  tetanus  toxin. 

Since  the  toxins  cannot  be  isolated  in  a  chemically  pure  form, 
their  exact  composition  cannot  be  known,  except  by  studying 
their  effects  upon  animals  and  animal  tissues.  Hence,  when  anti- 
toxin, added  to  toxin  in  a  test-tube  is  injected  into  an  animal, 
and  no  harm  results,  it  is  rightly  assumed  that  the  toxin  is 
neutralized,  and  both  are  chemically  bound;  yet  if  fresh  toxin  is 
added  to  the  mixture,  it  is  no  longer  neutral. 

If  the  toxin  of  the  pyocyaneus  and  the  anti-toxin  be  mixed  so 
that  they  neutralize  each  other,  and  if  the  mixture  is  heated,  the 
neutralization  disappears,  and  the  mixture  becomes  toxic  again. 
That  the  union  is  a  chemical  one,  may  be  inferred  from  the  fact 
that  it  is  more  rapid  in  concentrated  solution  than  in  weak,  and 
is  much  quicker  when  warmed  than  when  cold,  and  it  follows  the 
law  of  multiples,  i  part  toxin  neutralizing  i  part  of  anti- toxin, 
and  10  parts  of  toxin  neutralizing  10  parts  of  anti- toxin.  All 
this  is  in  accord  with  chemical  laws.  Toxins  sometimes  degener- 
ate into  what  Ehrlich  has  called  toxoids,  substances  that  bind 
(unite  with)  anti-toxin  just  as  effectively  as  toxins,  while  they 
are  not  poisonous,  yet  may  stimulate  healthy  cells  to  secrete 
anti- toxins  if  they  are  injected  into  the  body  of  experiment 
animals. 

More  is  known  about  the  toxins  of  diphtheria  and  tetanus  bacilli 
than  of  any  other.  Diphtheria  toxin  has  numerous  component 
substances,  one  of  which  is  the  toxin  that  causes  the  acute  phe- 
nomena of  diphtheria  intoxication.  Another,  toxon,  causes 
cachexia  and  paralysis  some  time  after  infection. 


44  INFECTION 

Tetanus  toxin  is  composed  of  two  substances:  tetanospasmin 
and  tetanolysin.  The  first  unites  chemically  with  the  motor  ele- 
ments of  the  nervous  system,  producing  degeneration  and  causing 
tremendous  contractions  of  the  muscles  governed  by  the  nerves 
involved.  The  second  has  the  property  of  dissolving  tissue,  such 
as  blood  cells. 

Tetanus  toxin  travels  from  the  infected  site  to  the  cord  by 
way  of  the  nerves;  it  is  exceedingly  poisonous;  a  single  prick  of 
the  finger  with  a  needle  moistened  with  toxin,  has  induced  tetanic 
symptoms. 

If  tetanus  toxin  of  known  strength  is  mixed  in  a  test-tube  with 
fresh  brain  substance  of  a  guinea  pig,  the  toxin  is  no  longer  toxic 
for  guinea  pigs.  This  shows  that  there  is  a  chemical  union  of 
the  toxin  and  the  cells  of  the  brain.  Cells  of  other  organs  have 
no  such  effect.  This  explains  specific  action  of  tetanus  upon 
nervous  tissue. 

Aggressins. — If  tubercle  bacilli  are  injected  into  the  abdominal 
cavity  of  a  guinea  pig,  tuberculosis  is  produced.  If  the  exudate 
produced  in  the  peritoneum,  consisting  of  fluid  and  cells,  be 
sterilized  and  injected  into  another  guinea  pig,  together  with 
some  virulent  tubercle  bacilli,  the  animal  will  succumb  in  twenty- 
four  hours.  If  the  exudate  alone  be  injected  no  effect  will  follow; 
if  bacilli  alone  are  injected,  a  tuberculous  peritonitis  will  be 
produced  in  a  few  weeks.  It  is  the  exudate  plus  bacilli  that 
does  the  harm.  The  exudate  is,  in  this  instance,  the  aggressin. 
Bail,  who  originated  the  doctrine  of  aggressins,  believes  that  a 
bacteriolysin  is  produced,  which,  acting  on  the  bacilli,  liberates 
an  endotoxin,  which  paralyzes  the  polynuclear  leucocytes, 
inhibiting  their  action  as  phagocytes. 

By  heating  the  exudate  to  6o°C.  the  aggressins  are  increased  in 
activity,  and  it  has  been  found  that  small  amounts  are  relatively 
stronger  than  larger  ones. 

This  phenomenon  has  been  explained  by  Bail  in  this  way.  He 
assumes  that  there  are  two  substances  in  the  exudate,  one  is 


AGGRESSINS  45 

thermolabile,  which  prevents  rapid  death,  the  other  is  thermo- 
stabile  and  this  is  favorable  to  rapid  death. 

Bail  assumes  that  a  tuberculous  cavity  in  an  animal  contains 
a  great  amount  of  the  aggressin,  which  prevents  chemotaxis  of 
the  poly  nuclear  leucocytes,  but  no.t  of  the  mononuclears  or 
lymphocytes. 

In  the  peritoneal  cavity  into  which  tubercle  bacilli  without 
aggressins,  have  been  injected,  an  active  phagocytosis  at  once  is 
begun  by  the  polynuclears,  and  the  injected  bacilli  are  in  a  great 
measure  destroyed,  and  those  left  develop  more  slowly,  producing 
a  tuberculosis  in  normal  course  of  time.  It  is  possible  to  im- 
munize animals  against  this  aggressin  producing  an  anti-ag- 
gressin,  which  substance  will  not  only  neutralize  the  aggressin 
but  also  stimulate  the  leucocytes  to  phagocytosis. 

This  aggressin  theory  has  been  applied  to  other  infections  with 
like  results,  notably  in  pneumococcus,  typhoid,  dysentery,  and 
plague  infection. 


CHAPTER  IV 


IMMUNITY 

By  immunity  is  understood  the  inherent  power  of  a  living  body 
to  successfully  withstand  the  invasion  of  infective  agents,  e.g., 
bacteria,  or  such  deleterious  and  toxic  substances  as  toxins,  drugs, 
complex  poisonous  albumins,  snake  venom,  foreign  blood  sera,  etc. 

The  following  tables  will,  perhaps,  be  helpful  in  the  study  of  the 
subject. 

Racial  immunity 


I.  Immunity 


2.  Inmiuiiity 


Natural 


Acquired 


Inherited  immunity 
Active  immunity 
Passive  immunity 

f  Anti-toxic 

I  Anti-bacterial 


It  is  a  well  known  fact  that  one  attack  of  an  infectious  disease 
generally  protects  an  individual  against  a  subsequent  attack.  It 
has  also  been  known  for  centuries  that  the  human  system,  by  first 
taking  very  small  doses,  and  gradually  increasing  them,  can  be  so 
accustomed  to  poison,  that  large,  and  otherwise  deadly  quantities 
may  be  taken  at  one  time  with  impunity.  Among  the  poisonous 
substances  to  which  men  can  accustom  themselves  are:  tobacco, 
morphia,'  arsenic,  and  alcohol.  Animals  treated  in  a  like  manner 
also  become  immunized  to  powerful  toxins,  snake  venom,  etc. 

Natural  Immunity. — The  hog  is  immune  to  snake  venom;  the 
chicken  to  tetanus.  Man  is  immune  to  hog,  or  chicken  cholera. 
The  negro  is  not  so  susceptible  to  yellow  fever  as  is  the  white. 
Animals  cannot  be  infected  with  scarlet  fever,  malaria,  and 
measles.  Young  adults  are  more  susceptible  to  typhoid  fever 
than  are  elderly  ones.  Infants  are  exceedingly  prone,  to  suffer 
from  milk  infection  while  older  children  are  not.  Again,  one 

46 


PHAGOCYTOSIS  47 

individual  may  contract  a  disease,  while  another  exposed  at  the 
same  time  will  not.  Inherited  immunity  is  exemplified  by  the 
history  of  races  into  which  a  new  disease  was  introduced  at  first 
with  high  mortality  but  later  with  great  reduction  in  the  severity 
of  the  infection. 

Acquired  Immunity. — Actively  acquired  by  infection.  One 
attack  of  yellow  fever  immunizes  the  individual  against  subse- 
quent attacks.  Vaccination  actively  immunizes  against  small-pox. 

Passively  Acquired. — Actually  injecting  protective  substances 
(anti-toxic  sera)  into  the  blood.  The  immunity  againsta  given 
disease  (diphtheria)  resides  in  the  anti-toxic  sera. 

Immunity  is  nearly  always  relative.  A  small  quantity  of  toxin 
may  be  innocuous,  while  a  large  quantity  may  cause  a  fatal 
toxaemia. 

There  have  been  several  theories  advanced  to  account  for  the 
various  phenomena  of  immunity,  the  oldest  ones,  beginning  with 
Pasteur,  being  that  some  substances  vitally  necessary  to  the 
virus  was  used  up  or  that  something  was  retained  to  affect  new 
microbial  attacks. 

The  modern  conception  of  immunity  deals  with  two  theories, 
the  theory  of  phagocytosis  of  Metchnikoff,  which  may  be  termed 
the  cellular  or  biologic  one,  and  the  lateral-chain,  or  the  humoral 
or  chemical  theory  of  Ehrlich.  Both  of  these  are  extremely 
ingenious  and  explain  satisfactorily  why  certain  bacteria  are 
unable  to  infect  the  body,  and  why,  the  body  once  infected,  cannot, 
in  many  diseases,  be  again  infected.  Furthermore  these  theories 
make  it  clear  to  us  why  the  body  tissues  during  life  do  not  fall 
an  easy  prey  to  many  putrefactive  bacteria,  as  after  death. 

Phagocytosis  is  essentially  a  theory  of  cell-devouring.  Leuco- 
cytes which  are  white  mobile  cells  of  the  blood,  and  other  fixed 
cells,  defend  the  body  against  infection  by  devouring  the  in- 
vading agents  of  disease  (Fig.  14). 

Metchnikoff  considers  the  subject  of  phagocytosis  under  three 
aspects;  (i)  nutritional;  (2)  resorptive;  (3)  protective. 


48  IMMUNITY 

Amoeba  and  certain  other  unicellular  vegetable  organisms 
belonging  to  the  myxomycetes  possessing  amoeboid  properties  and 
having  the  faculty  of  throwing  out  pseudopodia  or  protoplasmic 
arms,  acquire  their  food  by  enveloping  smaller  organisms  and 
other  nutritious  matter  which  they  absorb.  Certain  intracellular 
ferments,  which  they  possess,  digest  fibrin  and  gelatine,  and  con- 
vert starch  into  sugar.  These  cells  protect  themselves  against 
inimical  microorganisms  by  enveloping  and  digesting  them. 


FIG.  14. — Phagocytosis.     Gonococci  in  leucocytes  in  pus  from  gonorrhoea. 
(Kolle  and  Wassermann.) 

They  are  attracted  by  food  and  moisture  (called  positive  chemo- 
taxis)  and  repelled  by  strong  solution  of  salt,  poisons,  etc.  (nega- 
tive chemotaxis) . 

Higher  in  the  animal  scale  among  the  multicellular  organisms, 
the  cells  of  the  intestines  have  the  property  of  absorbing  and  di- 
gesting food.  These  fixed  cells  are  called  sessile  phagocytes. 
Still  higher  in  the  scale  (man)  certain  digesting  cells  are  present 
in  the  digestive  tract,  which  are  incapable  of  absorbing  food. 
They,  however,  secrete  ferments  which  digest  gelatine  and  fibrin, 
and  convert  starch  into  sugar.  But  other  cells  of  the  animal ' 
body,  the  leucocytes,  large  mononuclears  and  certain  fixed  tissues- 
cells,  have  the  property  of  engulfing  foreign  bodies  like  bacteria 


PHAGOCYTOSIS  49 

and  of  digesting  them;  it  is  to  these  that  Metchnikoff  ascribed 
the  protective  power  of  phagocytosis.  Cells  of  a  protecting 
character  in  man  are  either  microphages  or  macrophages.  The 
microphages  are  the  polynuclear  leucocytes,  which  are  concerned 
in  the  protection  of  the  organism  against  acute  infections,  the 
bacteria  of  which  they  take  up  and  devour.  The  macrophages 
consist  of  the  large  lymphocytes,  the  endothelial  cells,  and  some 
connective  tissue  cells,  which  take  up  foreign  bodies.  Both  of 
these  classes  contain  ferments;  microcytase  being  found  in  the 
microphages;  and  macrocytase  in  the  macrophages.  The  latter 
absorb  connective  tissue  cells  through  their  particular  ferments, 
and  are  active  in  immunizing  against  tuberculosis.  These  cells 
perform  various  functions  in  the  body.  When  the  tissues  are 
invaded  with  bacteria,  the  blood  shows  an  increase  in  the  number 
of  these  microphages,  which  have  been  called  the  "hygienic 
police."  Summoned  to  repel  invasion,  they  leave  the  lymph 
stream  for  that  of  the  blood.  All  the  phenomena  of  leucocytic 
emigration  in  inflammation  is  a  manifestation  of  positive 
chemo taxis.  During  practically  all  the  infections,  the  peripheral 
blood  contains  an  excess  of  leucocytes  over  the  normal  amount 
per  cubic  millimeter  (7,600).  In  exceptional  infections,  typhoid 
fever,  influenza,  measles,  and  tuberculosis,  there  is  no  such 
increase,  or  leucocytosis.  In  malaria  (not  a  bacterial  infection) 
there  is  also  no  leucocytosis. 

Metchnikoff  has  described  a  process  in  which  the  phagocytes 
undergo  what  he  calls  phagolysis.  The  ferment,  cytase,  is  dis- 
charged and  acts  extracellularly,  digesting  the  cell  body  and 
freeing  ferment  which  will  act  against  the  invaders.  Metchnikoff 
further  claims  that  both  phagocytosis  and  phagolysis,  either 
severally,  or  in  combination,  are  responsible  for  natural  or  ac- 
quired immunity. 

In  the  case  of  acquired  immunity,  it  is  supposed  that  the  leuco- 
cytes become  educated.  Regarding  the  toxins  against  which 
animals  can  be  immunized  by  gradually  increased  doses,  it  is  held 


50  IMMUNITY 

by  him  that  the  educated  leucocytes  neutralize  the  poison  by 
their  secretions. 

In  the  case  of  anthrax  infection,  animals  infected  with  virulent 
cultures  of  this  organism  quickly  succumb,  without  exhibiting 
any  leucocytosis  (negative  chemo taxis). 

If  the  animal  has  been  previously  immunized  with  attenuated 
culture  the  injection  of  a  virulent  culture  is  followed  by  an  enor- 
mous outpouring  of  leucocytes  at  the  site  (positive  chemo  taxis), 
while  if  the  site  of  the  inoculation  in  the  non-immune  animal  is 
examined,  only  a  few  leucocytes,  and  some  clear  serum  will  be 
found. 

Toxins,  if  injected,  cause  a  negative  chemotaxis.  If  tetanus 
spores  are  injected  into  an  animal,  together  with  some  toxin,  the 
animal  rapidly  succumbs  to  tetanus,  without  evincing  any  leuco- 
cytosis. If  the  spores  are  washed  free  from  toxin,  and  injected, 
active  leucocytosis  occurs  and  the  animal  survives. 

A  mixed  infection  of  a  highly  virulent  culture,  and  a  non- 
virulent  one,  often  hastens  the  action  of  the  virulent  one.  It 
is  supposed  that  the  non-virulent  bacteria  engage  the  leucocytes, 
so  that  these  cells  cannot  cope  with  the  virulent  ones. 

Phagocytosis  thus  plays  an  important  part  in  a  protective  role 
in  natural  immunity,  but  no  satisfactory  theory  has  yet  been 
offered  in  explanation  of  the  protective  process  in  acquired 
immunity,  at  least  against  toxins  and  other  soluble  and  unorgan- 
ized poisons. 

In  order  to  meet  the  criticisms  arising  after  Ehrlich's  theories. 
Metchnikoff  added  to  his  theory  by  stating  that  complement 
and-anti-body  are  enzymic  substances  derived  from  phagocytes. 

The  cellulo-humoral  theory  claims  the  attention  of  most  bac- 
teriologists, as  the  probable  explanation  of  the  phenomena  of 
immunity.  It  is  certain  that  cells,  either  sessile  or  mobile,  and 
fluids,  are  important  means  of  internal  defense.  In  order  that ' 
this  theory  may  be  comprehended,  certain  well-known  properties 
on  formal  and  artificially  immunized  serum  must  be  understood. 


NATURAL   IMMUNE   BODIES  51 

Alexins. — It  has  been  found  by  numerous  observers,  that  nor- 
mal  blood  serum  is  germicidal  for  many  bacteria,  and  pecu- 
liarly active  substance  that  is  contained  in  the  serum,  was  called 
by  Buchner  Alexin.  This  dissolves  bacteria  and  destroys  them. 
It  also  destroys  the  red  blood  cells  of  other  animals.  The  alexin 
of  a  dog's  serum  dissolves  the  red  cells  of  a  rabbit;  it  is  therefore 
hamolytic.  It  also  is  thermolabile,  that  is,  its  properties  are 
destroyed  by  heat  (55°C.).  It  is  identical  with  the  complement 
of  Ehrlich,  and  the  cytase  of  Metchnikoff. 

The  complement,  as  it  will  hereafter  be  called,  takes,  as  already 
stated,  an  active  part  in  bacteriolysis,  or  bacteria-dissolving,  and 
in  haemolysis,  or  blood-dissolving,  it  is  present  hi  normal  non- 
immune  sera.  R.  Pfeiffer  found  that  if  some  serum  from  a  guinea 
pig  immunized  against  cholera  spirilla  is  injected  into  the  peri- 
toneal cavity  of  a  healthy  non-immune  guinea  pig,  with  some 
cholera  spirilla,  that  the  latter  are  agglutinated,  and  ultimately 
dissolved,  having  undergone  bacteriolysis  (Pfeiffer's  reaction). 
The  immune  serum  alone  in  a  test-tube,  with  the  cholera  spirilla 
does  not  have  this  action,  but  if  some  normal  guinea-pig  serum 
is  added  to  the  mixture,  an  immediate  solution  takes  place, 
showing  that  the  presence  of  both  the  normal  serum  containing 
the  complement,  and  the  immune  serum,  containing  the  immune 
body,  or  amboceptor,  are  necessary  to  complete  the  solution  of 
the  bacteria.  If  the  complement  is  heated  above  55°C.  for  an 
hour,  solution  does  not  take  place,  even  if  the  immune  serum  is 
present,  but,  after  heating  the  mixture,  it  may  be  reactivated  by 
adding  some  fresh  unheated  complement.  The  complement  is 
thermolabile,  i.e.,  destroyed  by  heat. 

The  immune  serum  is  not  affected  by  heat,  and  is  therefore 
called  thermostabile. 

These  various  reactions  may  be  expressed  concretely  thus: 

Bacteria  +  immune  body  =  no  solution. 
Bacteria  +  complement  =  no  solution. 


52  IMMUNITY 

Bacteria +inimune  body  +  complement  =  solution  (Pfeiffer's 
reaction). 

Bacteria  +  immune  body  +  complement  (heated)  =  no  solu- 
tion. 

Bacteria  +  immune  body  (heated)  +  complement  =  solution. 

The  same  phenomena  have  been  observed  in  the  blood  of 
animals  immunized  against  the  red  blood  corpuscles  of  another 
animal  of  foreign  species. 

If  a  rabbit  is  immunized  with  the  blood  of  a  dog  by  repeated 
and  increasing  doses,  the  serum  of  that  rabbit  will  become  h&mo- 
lytic  to  the  corpuscles  of  the  dog's  blood  if  they  are  mixed,  pro- 
vided some  normal  rabbit's  blood  complement  is  added  to  the 
mixture. 

Dog's   erythrocytes  +  immune   rabbit   serum  =  no   solution. 
Dog's   erythrocytes  +  immune  rabbit  serum  +  complement  = 
solution. 

Dog's  erythrocytes  +  immune  rabbit  serum  +  complement, 
heated  =  no  solution. 

The  immune  body  acts  as  a  preparer  of  the  corpuscles,  or 
bacteria,  so  that  the  complement  can  act  upon  the  cells.  The 
reaction  is  very  like  the  action  of  pepsin  on  fibrin.  Hydrochloric 
acid  must  be  present. 

(1)  Pepsin  +  fibrin  =  no  solution  or  lysis. 

(2)  HC1  +  fibrin  =  no  solution  or  lysis. 

(3)  Pepsin  +  HC1  +  fibrin  =  solution  or  lysis. 
The  HC1  corresponds  to  the  immune  body. 

In  the  case  of  haemolysis,  or  bacteriolysis  the  action  of  the  im- 
mune body  is  specific.  The  immune  body  of  cholera  spirilla  will 
not  prepare,  or  fix  typhoid  bacilli,  so  that  they  can  be  acted  upon  by 
the  complement.  Nor  will  the  immune  body  of  dog's  erythrocytes 
prepare  those  of  a  pig,  so  that  the  complement  may  act  on  themu 

A  loose  chemical  union  takes  place  between  the  bacteria  and 
the  immune  body,  but  no  such  union  occurs  between  the  comple- 


AGGLUTININS  53 

ment  and  the  bacteria.  The  same  chemical  union  occurs  between 
the  red  cells  and  the  immune  body  in  haemolysis,  but  not  between 
the  cells  and  the  complement. 

Ehrlich  holds  that  there  are  many  complements,  each  one  dif- 
ferent from  the  other,  and  that  their  action  is  specific  for  the 
different  kinds  of  bacteria  or  cells  with  which  an  animal  may  be 
immunized.  Bordet  and  Buchner,  on  the  other  hand,  maintain 
that  there  is  but  one  complement. 

The  solution  of  any  cells  by  immune  bodies,  or  anti-bodies,  as 
they  have  been  called,  is  known  as  cytolysis.  And  cytolysins 
may  be  produced  by  making  anti-bodies  of  nerve  cells,  leucocytes, 
epithelial  cells,  liver  cells,  as  well  as  blood  cells,  by  immunizing 
an  animal  against  these  different  cells  with  repeated  injections 
of  the  cells  or  emulsions  of  them. 

Agglutinins  are  peculiar  bodies  which  have  the  property  of 
causing  certain  cells  to  agglutinate.  One  of  the  earliest  manifes- 
tations of  immunity  of  a  certain  serum  to  bacteria,  or  to  blood 
cells,  is  this  peculiar  action  of  the  serum  causing  either  the  bac- 
teria or  blood  cells  to  clump  together  in  masses.  Part  of  Pfeiffer's 
reaction  is  the  agglutination  of  the  cholera  spirilla  in  clumps 
before  they  are  dissolved  by  the  complement  and  immune  body. 

Recent  studies  accord  to  agglutinins  one  of  the  most  impor- 
tant places  in  the  defense  against  disease.  Their  action  is 
believed  to  facilitate  that  of  lysins  and  opsonins,  helping  the 
latter  by  fixing  a  group  of  bacteria  so  that  they  may  be  the 
better  prepared  for  phagocytosis. 

If  the  serum  of  a  typhoid  fever  patient  is  mixed,  even  in  high 
dilutions  with  some  typhoid  bacilli,  the  latter  are  clumped  in 
isolated  groups.  Clinically  this  is  known  as  the  Widal  reaction, 
and  is  the  most  reliable  single  sign  of  typhoid  fever. 

These  agglutinins  may  be  produced  artificially  by  injecting 
large  and  increasing  doses  of  bacteria  into  animals.  After  a  time, 
in  the  serum  of  the  rabbit,  there  develops  a  peculiar  body  which 
agglutinates  typhoid  bacilli,  if  they  are  brought  in  contact  with 


54  IMMUNITY 

it.  Sera  can  be  rendered  so  highly  agglutinative  as  to  produce 
this  reaction  even  if  diluted  100,000  times  or  more. 

If  an  animal  is  immunized  against  spermatozoa,  or  the  red 
blood  cells  of  a  foreign  species,  its  serum  becomes  agglutinative 
to  these  cells. 

Precipitins. — If  a  rabbit,  or  any  other  animal  in  fact,  is  immun- 
ized by  repeated  injections  of  foreign  protein  (blood,  bacterial 
culture,  etc.)  peculiar  bodies  develop  in  its  blood  serum  called 
precipitins,  and  these  can  be  demonstrated  by  adding  to  the 
serum  of  the  immunized  animal  in  a  test-tube  a  minute  portion 
of  the  material  against  which  the  animal  was  immunized.  As 
soon  as  the  immunized  serum  and  the  specific  substance  are 
mixed,  a  precipitate  forms.  This  is  another  phenomenon  of 
immunity,  and  is  of  more  than  theoretical  importance  in  medicine. 
The  reaction  is  strictly  specific;  thus,  if  the  serum  of  a  goat  is 
injected  into  a  rabbit  repeatedly  the  rabbit's  blood  will  form  a 
precipitate  with  normal  goat's  serum  if  the  two  are  mixed  in 
a  test-tube.  Old  dried  blood,  semi-putrid  blood,  blood  on  white- 
wash, or  rusty  steel,  even  in  minute  quantities,  if  dissolved  in  salt 
solution,  may  be  used  to  produce  this  reaction.  In  medico-legal 
matters,  this  test  is  of  use  for  the  identification  of  human  blood. 
By  some,  the  phenomenon  of  agglutination  is  supposed  to  be  due 
to  the  formation  of  a  precipitin,  in  the  meshes  of  which  bacteria  or 
blood  cells  are  caught  and  agglutinated,  and  that  agglutination  is 
but  a  modification  of  the  formation  of  precipitins. 

Anti-toxin  formation  is  also  another  phenomenon  of  immunity. 
If  an  animal,  such  as  a  horse,  receives  numerous  increasing  doses 
of  a  given  toxin,  say  that  of  tetanus,  it,  in  a  short  time,  becomes  so 
accustomed  to  the  poison,  that  it  can  withstand  the  administration 
of  immense  doses.  (If  these  large  doses  had  been  given  at  first, 
they  would  have  proved  fatal.)  If  the  horse  is  then  bled,  and  its 
serum  injected  into  rabbits  or  guinea  pigs,  they  may  receive 
shortly  after,  at  one  dose,  enough  toxin  to  kill  ten  such  animals. 
The  horse  serum  thus  protected  these  animals  against  the  toxin, 
as  it  was  anti-dotal,  or  in  other  words  anti-toxic.  A  chemical 


LATERAL   CHAIN   THEORY  55 

union  occurs  between  the  toxin  and  the  anti-toxin,  since,  according 
to  the  law  of  multiples,  a  definite  amount  of  anti-toxin  unites 
with  a  definite  amount  of  toxin.  If  ten  times  the  amount  of 
anti-toxin  is  used  it  will  exactly  neutralize  ten  times  the  amount 
of  toxin,  and  the  mixture  becomes  inert.  Again,  the  union  of 
the  two  substances  follows  well-known  chemical  laws,  whereby 
chemical  union  takes  place  more  rapidly  in  concentrated  than  in 
dilute  solutions,  and  when  the  solutions  are  warm.  If  the  mixture 
of  toxin  and  anti-toxin  is  heated,  it,  instead  of  being  neutral, 
becomes  toxic  again.  This  toxicity  can  be  neutralized  again  by 
the  addition  of  fresh  unheated  an ti- toxic  serum  (reactivation). 

The  production  of  bacteriolysins,  cytolysins,  agglutinins,  pre- 
cipitins,  and  anti-toxins  are  manifestations  of  the  activity  of  the 
immunized  organisms.  To  further  understand  this  activity, 
Ehrlich's  side-chain  theory  of  immunity  must  be  comprehended. 
This  is  known  as  the  chemical  theory.  To  understand  it  fully 
some  consideration  must  be  given  to  the  study  of  the  toxin  mole- 
cule. Ehrlich  believes  that  each  molecule  of  toxin  is  made  up  of 
two  groups  of  atoms,  constituting  what  is  known  in  chemical 
nomenclature  as  lateral  chains.  Many  molecules  are  made  up 
of  a  central  body  and  lateral  chain  of  atoms  which  are  free  to 
combine  with  other  groups  of  atoms  without  disturbing  the  central 
body. 

The  benzol  ring  is  very  suitable  for  the  demonstration  of  the 
relationship  of  the  side  chain  to  the  central  body. 

H 
I 

/C\ 
H— C        ^C— H 

II  I 

H— ( 


H 

BENZOL. 


56  IMMUNITY 

The  benzol  molecule  CeH6  is  here  represented  graphically  as  a 
ring  with  a  central  nucleus  of  C6  with  lateral  chains  of  H.  con- 
necting each  atom  of  C. 

If  one  of  these  lateral  chains  H.  is  supplanted  by  the  acid  radical 
COOH.  the  benzol  is  converted  into  benzoic  acid  and  its  formula  is 
represented  thus: 

O 

y 

C— OH 


H— C       ^C— H 


H—  C          C—  H 


H 

BENZOIC  ACID. 

If  to  this  acid  radical  of  the  benzoic  ring,  sodium  hydroxid 
unites,  supplanting  an  H  in  the  OH  of  this  radical,  we  have, 
instead  of  benzoic  acid,  benzoate  of  soda.  . 

O 

y 

C—  O—  Na 

/\ 
H—  C      C—  H 

II       I 
H—  C      C—  H 

\y 

C 

I 

H 

BENZOATE  OF  SODA. 


RECEPTORS  57 

It  is  thought  that  as  the  soda  is  brought  in  contact  with  the 
central  nucleus  of  the  benzol  ring,  so  foodstuffs  unite  with  the 
central  body  of  the  cell  molecule  in  the  organism  and  nourish  it. 

In  the  case  of  toxin,  the  two  lateral  chains  of  its  molecule  are 
called  haptophores  and  toxophores.  The  haptophores  seize  the 
lateral  chains  of  the  cell  and  the  toxophores  poison  it. 

Ehrlich  conceived  that  cells  were  nourished  by  their  lateral 
chains,  each  having  a  central  nucleus  with  many  lateral  chains 
called  receptors  bristling  all  over  it.  Complex  albumins,  food- 
stuffs or  poisons  (as  the  case  may  be)  unite  with  it.  This  means 
a  chemical  union  of  a  part  of  a  cell  with  all  or  part  of  a  group  of 
atoms.  But  certain  body  cells  are  only  capable  of  uniting  with 
certain  toxins.  It  is  known  that  the  toxin  of  tetanus  has  a  chem- 
ical affinity  for  the  nervous  system  and  for  its  neural  elements 
and  not  for  liver  or  spleen  cells.  The  poisons  of  snake  venom 
seem  incapable  of  uniting  with  any  cells  of  the  pig;  therefore,  this 
animal  is  immune  to  snake  venom. 

Now,  as  these  toxins  unite  with  the  cells  by  means  of  the  recep- 
tors, the  cell  is  stimulated  to  produce  an  excessive  number  of  these 
receptors,  which  are  cast  off  and  become  free.  Nature  is  very 
prodigal  and  whenever  any  of  the  tissues  of  the  body  have  been 
injured,  or  there  is  a  deficiency,  an  enormous  excess  of  reparative 
cells  is  produced.  Weigert  first  called  attention  to  this  phenome- 
non, which  has  been  called  Weigert's  overproduction  theory.  So 
when  the  haptophores  of  the  toxin  molecule  combine  with  the 
receptors  of  the  cell,  the  latter  are  incapable  of  any  further  union 
and  are  useless  to  the  cell.  Accordingly  a  great  number  of  free 
receptors  are  generated,  and  floating  in  the  blood,  engage  the 
haptophorous  portion  of  the  toxin.  Thus  the  toxophore  is  neu- 
tralized and  rendered  innocuous  before  it  can  reach  the  cell. 
These  free  overproduced  receptors  constitute  the  anti-toxin. 
This  is  the  essence  of  Ehrlich's  theory  (Fig.  15). 

Through  the  process  of  time  and  oxygenation  the  toxophorous 
group  in  the  toxin  becomes  innocuous,  and  only  the  haptophorous 


IMMUNITY 


group  remains  active;  nevertheless  the  haptophorous  group  is  able 
to  combine  with  the  receptors  and  to  stimulate  the  cell  into 
generating  free  receptors.  This  attenuated  toxin  is  called  by 
Ehrlich  toxoid.  The  receptors  have  been  compared  to  a  lightning 
rod,  which  if  placed  within  a  building  would,  if  struck,  cause 
disaster,  while  the  same  rod  placed  outside  of  the  building,  is  a 
means  of  protection  to  the  structure  against  lightning.  This 


FIG.  15. — a,  receptor  on  cell;  b,  toxin  molecule;  c,  haptophorous  portion  of 
the  molecule;  dy  toxophorous  portion;  e,  receptor.     (Williams.) 

theory  can  be  applied  to  the  production  of  other  anti-bodies. 
If  blood  cell,  bacterial  cell,  or  any  animal  fluid  possessing  a  hapto- 
phore  is  capable  of  combining  with  side  chains  (receptors)  of  the 
cells  of  the  immunized,  just  as  a  key  fits  a  lock,  then  the  cells  are 
stimulated  to  produce  excessive  numbers  of  receptors,  and  these 
constitute  the  anti,  or  immune  body.  It  is  possible  to  produce 
from  rennet,  egg-albumin,  cow's  milk,  and  from  many  other- 
albuminous  substances,  immune  bodies  by  injecting  these  sub- 
stances into  animals  (Figs.  16,  17). 


IMMUNE   BODIES 


59 


FIG.  16.— EHRLICH'S  LATERAL-CHAIN  THEORY.  Cell  with  num- 
>us  receptors  of  various  kinds  and  shapes  to  which  are  united  the  toxin 
jlecule.  Note  the  free  receptors. 


FIG.  17.— EHRLICH'S  LATERAL-CHAIN  THEORY.  In  one  figure  the 
free  receptors  (anti-bodies)  are  united  with  the  toxin  molecule,  the  attached 
receptors  have  no  haptophores  united  to  cell. 


6o 


IMMUNITY 


List  of  immune  bodies  and  their  anti-bodies  (Ricketts) 
Antigens  or     Products  of 
Immunizing    Immunization 
Substances 


Toxins 
Complements 

Ferments 
Precipitogenous 

Substances 
A  gglutinogenous 

Substances 
Opsinogenous 

Substances 
Cytotoxin  Produc 

ing  Substances 


Complement 

Alexin. 

Cytase 


Anti-toxins 
Anti-comple- 
ments 

Anti-ferments 
Precipitins 

Agglutinins 
Opsinins 
Cytotoxins . . . 


Uemolysins 
Bacteriolysins 
Special  cytotox- 

ins  Such  as 
Spermotoxin 
Nephrotoxin 
Hepatotoxin,  etc. 

Synonyms 

Amboceptor. 

Immunkb'rper. 

Zwischenkb'rper. 

Intermediary  body. 

Fixateur. 

Preparateur. 


Consisting  of 
two  bodies 
Complement 
Amboceptor 


Desmon. 

Substance  sensibilisatrice. 

It  is  well  known  that  rennet  coagulates  milk,  but  if  some  of  the 
serum  of  an  animal  immunized  against  rennet  is  added  to  the  milk, 
the  latter  cannot  be  coagulated  because  the  anti-rennin  combines 
with  the  rennet  and  renders  it  inert. 


IMMUNE  BODIES  6 1 

The  production  of  bacteriolysins  is  explained  by  Ehrlich's  lat- 
eral-chain hypothesis.  Immunization  against  bacteria  which  do 
not  produce  soluble  toxins  is  easily  secured  by  repeated  injection 
of  either  dead  or  living  bacteria  into  the  organism.  It  is  not  easy, 
however,  to  confer  passive  immunity,  as  in  the  case  of  diphtheria, 
by  the  injection  of  the  serum  of  the  immunized  animal.  The 
immune  body  is  alone  present  in  the  serum  generally  and  some 
complement  must  be  added  to  effect  bacteriolysis.  The  serums 
which  aid  in  the  solution  of  bacteria  are  known  as  anti-bacterial 
serums,  which,  though  not  anti-toxic,  may  check  invasions  and 
aid  in  recovery  by  destroying  bacteria.  It  is  possible  to  effect 
an  in  corpore  bacteriolysis  in  the  case  of  typhoid  fever  if  the 
immune  body  and  complement  are  injected  in  sufficient  amounts 
and  proportions.  As  yet  the  results  are  not  satisfactory  from  a 
clinical  standpoint. 

A  study  of  Fig.  18  will  show  clearly  the  exact  combinations  of 
various  substances  engaged  in  the  immunity  process.  Some  of 
the  terms  must  be  defined. 

Antigen,  the  body,  bacterium,  red  blood  cell,  etc.,  used  for 
stimulating  the  production  of  thermostabile  anti-bodies,  which 
latter  are  then  the  substances  formed  against  antigens;  inciting 
substance-antigen. 

Toxins,  ferments,  see  above. 

Toxophore,  the  poison-carrying  fraction  of  the  antigen. 

Haptophore,   the  binding  fraction  of  antigen  or  anti-body. 

Complement,  alexin  the  normal  thermolabile  anti-body  sub- 
stance in  serum. 

Zymophore,  toxophore  for  agglutinins  and  precipitins. 

Cytophile  fraction  is  that  part  of  anti-body  which  combines 
with  cell,  while  complement phile  fraction  joins  with  complement. 

Immune  body,  the  thermostabile  anti-body  against  bacterial  or 
other  cells. 

By  immunizing  with  complement  or  anti-body  we  obtain 
respectively  anti-complement  and  anti-immune  body  which 


62 


IMMUNITY 


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ANAPHYLAXIS  63 

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experimentally  will  neutralize  the  action  of  these  two  substances. 
The  complement  being  the  really  responsible  potent  factor  in  all 
these  reactions  it  may  be  assumed  to  have  two  binding  affinities, 
one  to  the  cells  which  it  designs  to  help  and  another  effect  upon 
antigen.  If  the  former  be  absorbed  in  any  abnormal  manner 
the  latter  is  valueless. 

Cell  Receptor  and  Immune  Bodies  (follow  Fig.  18). — First  Order: 
I  Simple  union  of  toxins  (soluble)  and  fixed  or  free  receptors  or  antil 
!  toxins;  no  complement  needed. 

Second  Order :  Concerns  agglutination  and  precipitation.  Anti- 
gen has  two  affinities,  one  for  the  haptophore  of  anti-body,  another 
for  the  agglutinin  of  the  anti-body.  The  anti-body  must  therefore 
have  reversed  corresponding  fractions.  The  zymophore  of  anti- 
body acts  when  the  two  haptophores  have  united  and  produces 
the  agglutination  or  precipitation.  No  complement  is  needed. 

Third  Order :  Concerns  bacteriolysins,  hemolysins  or  bacterioly- 
sins,  etc. ;  have  haptophore  for  anti-body,  and  a  toxophore.  Anti- 
body has  haptophore  for  antigen  and  for  the  haptophore  of  the 
complement.  The  union  of  the  three  must  occur.  Complement 
is  necessary  for  the  destruction  of  the  bacteria  which  it  accom- 
plishes through  its  zymophore. 

Anaphylaxis. — Against  protection,  the  opposite  of  prophylaxis; 
also  called  Hypersusceptibility.  This  phenomenon,  first  de- 
scribed by  Theobald  Smith,  Portier  and  Richet,  consists  in  a 
condition  of  extreme  sensitiveness  of  animals  against  foreign  pro- 
teins. If  a  guinea  pig  be  injected  into  the  peritoneum  with  a 
minute  quantity,  say  M>000  gram>  of  horses'  serum  and  eight  to 
ten  days  later  receive  a  quantity  of  Ko  gram>  the  animal  will  be- 
come uneasy,  then  depressed,  have  dyspnea,  scratch  itself  vio- 
lently about  the  face  and  finally  die  after  an  intensification  of 
these  symptoms.  Similar  symptoms  have  been  observed  in  per- 
sons receiving  diphtheria  anti-toxin  therapeutically.  The  condi- 
tion of  high  sensitivity  to  this  anti-toxin  is  called  allergie  and 
upon  its  degree  depends  the  reaction  following  anti-toxin  admin- 


64  IMMUNITY 

istration.  The  skin  eruptions,  joint  pains  and  edema  of  serum 
sickness  are  also  evidences  of  this  condition.  It  is  said  that  those 
persons  who  suffer  after  anti-toxin  are  susceptible  to  the  emana- 
tions from  horses  and  the  physician  should  make  inquiries  in  this 
direction  when  contemplating  the  injection  of  all  sera. 

In  experimentally  induced  hypersusceptibility  the  reaction  is 
specific.  The  condition  is  transmissible  from  mother  to  foetus 
and  it  can  be  transferred  from  adult  to  adult  passively  by  injecting 
the  blood  of  a  sensitive  animal  into  a  normal  one.  The  first  dose 
is  called  the  sensitizing  one,  the  second  the  intoxicating.  The 
incubation  period  of  the  sensitization  varies  with  the  nature  of  the 
protein;  for  horse  serum  it  is  from  eight  to  twelve  days,  for  bac-  | 
terial  proteins  from  five  to  eight  days.  The  sensitive  period  may  j 
last  for  several  years.  In  searching  for  the  cause  of  this  reaction 
it  was  found  that  there  are  (i)  a  spastic  distention  of  the  pul- 
monary alveoli  probably  both  of  central  and  local  nature,  (2) 
scattered  hemorrhages  in  the  organs  and  (3)  hemorrhages  with 
ulcerations  in  the  gastric  mucosa.  There  have  been  many  theories 
for  this  phenomenon,  but  those  of  Vaughan,  Friedberger  and  Wolff 
Eisner  may  be  condensed  and  compounded  about  as  follows.  The 
body  is  unprepared  to  care  for  parenterally  (otherwise  than  gastro- 
intestinal tract)  introduced  protein  and  must  develop  anti-body 
or  enzyme  to  care  for  it.  This  enzyme  or  anti-body  works  slowly 
and  carefully  disposes  of  the  foreign  protein,  the  products  of  which 
are  slowly  absorbed  and  removed.  In  accord  with  the  overpro- 
duction theory  this  anti-substance  is  in  large  quantity  when  an- 
other introduction  of  protein  occurs,  and  goes  to  its  work  with 
avidity  so  that  it  rapidly  breaks  the  protein  up  into  toxic  elements 
which  cannot  suddenly  be  cared  for  by  the  body.  It  is  also 
thought  by  some  workers  that  the  body  protein  of  the  animal  in 
question  is  attacked  and  split,  liberating  toxic  fractions,  since  the 
injected  protein  would  not  be  adequate  in  amount  to  accomplish 
poisoning.  These  protein  toxins  attack  nervous  and  parenchy- 
matous  tissues.  Another  very  plausible  theory  would  have  it 


ANAPHYLAXIS  65 

that  the  first  injection  directly  sensitizes  important  cells  which  are 
destroyed  when  an  intoxicating  dose  arrives. 

The  similarity  of  characters  between  serum  shock  and  immedi- 
ate traumatic  shock  was  thought  to  shed  some  light  on  the  sub- 
ject. The  latter  is  believed  to  be  due  to  protein  degradation 
products,  due  to  trauma  or  low  blood  pressure,  which  have  a 
paralyzing  effect  upon  unstriped  muscle  or  upon  the  vasomotor 
centers. 

It  has  been  shown  that  an  anti-anaphylactic  state  can  be  pro- 
duced by  repeated  small  injections  of  protein  at  intervals  too 
short  to  allow  incubation  of  an  intoxicating  dose.  Use  is  made 
of  this  knowledge  in  the  case  of  persons  who  need  anti-serum  but 
who  show  sensitivity  to  it.  Repeated  small  but  increasing  quan- 
tities are  put  into  or  under  the  skin  or  into  the  vein  until  the 
patient  can  receive  without  reaction  the  full  dose.  This  is  called 
desensitization. 

Friedberger  has  used  these  facts  to  elaborate  a  theory  of  infec- 
tion. He  believes  that  bacteria  circulating  in  the  body  stimulate 
anti-bodies,  combine  with  them  and  that  when  complement  acts 
upon  this  union  toxic  substances  are  set  free. 

In  explaining  all  infectious  diseases  on  this  basis  one  assumes 
that  sometime  in  life  a  person  has  been  sensitized  by  bacteria  or 
their  proteins  so  that  he  is  receptive  for  a  virulent  germ  when 
this  has  overcome  the  primary  external  bodily  defenses.  It  is  also 
to  be  considered  the  modern  explanation  of  diathesis. 

McKail  divides  anaphylaxis  as  follows: 

Natural  Anaphylaxis,  depending  upon 

(a)  Species  of  animal,  for  example  cholera  in  man,  anthrax 

in  cattle,  glanders  in  horses. 

(£)  Age — diphtheria  in  children,  erysipelas  in  the  elderly. 
(c)  Individual — to  white  of  egg,  or  blood  serum,  even  by  in- 

gestion  ("one  man's  meat  is  another  man's  poison"), 

5 


66  IMMUNITY 

Acquired  Anaphylaxis,  depending  upon 

(a)  An  attack  of  disease,  erysipelas,  diphtheria. 

(b)  The  injection  of  dead  cells,  tuberculin. 

(c)  Injection  of  nitrogenous  matter,  blood  serum  and  egg- 
white. 

Direct  practical  application  of  theoretical  speculation  about 
hypersensitivity  may  be  made  in  explaining,  treating  and  prevent- 
ing certain  states  in  man.  Mention  has  already  been  made  of  the 
possibility  of  intoxicating  persons  susceptible  to  the  presence  of 
horses  by  the  injecting  of  horse  serum.  The  only  treatment  for 
such  a  "Schock"  is  an  injection  of  epinephrin  or  atropin. 

Serum  sickness  is  explained  as  a  digestion  of  foreign  serum  still  in 
the  body  as  such  when  sufficient  ferment  has  accumulated  to 
digest  it,  a  peroid  of  three  to  twelve  days.  Whether  this  be  cor- 
rect or  not,  symptoms  can  be  made  milder  or  prevented  by  dividing 
the  doses  by  some  hours.  A  state  of  hypersensitivity  or  allergic 
may  also  exist  to  pollen  of  plants,  certain  foods  and  drugs,  dusts 
of  feathers  and  hair  or  dandruff  from  cats  and  dogs.  The  evi-  j 
dences  of  this  state  take  the  form  of  asthma,  gastrointestinal  dis- ' 
turbances,  and  skin  eruptions  of  which  urticaria  and  eczema  are  i 
the  commonest.  Detection  of  this  hypersensitivity  may  be  sub-  | 
jective  at  times  but  usually  it  has  to  be  established  by  technical 
means.  It  so  happens  that  allergic  persons  have  either  a  definite 
anti-body  reaction  or  the  local  sessile  receptors  in  the  cutaneous 
cells  are  stimulated,  for  the  application  of  the  responsible  protein 
to  an  abraded  area  on  the  skin  will  produce  at  the  point  a  swollen 
red  areola.  In  the  cases  of  infection  of  a  bacterial  or  toxic  mix- 
ture this  reaction  usually  requires  twenty-four  hours  to  develop, 
a  delay  suggesting  that  anti-bodies  are  involved.  In  the  case  of 
serum  or  pollen  allergic,  the  reaction  is  almost  immediate,  suggest- 
ing local  cellular  preparation.  This  is  the  basis  of  skin  tests, 
except  with  tuberculin  and  the  S chick  test.  Prevention  of  such 
allergic  poisonings  is  best  accomplished  by  the  avoidance  of 


ANAPHYLAXIS 


67 


the  offending  material.  Treatment  and  prophylaxis  take  the 
form  of  injecting  solutions  of  the  responsible  protein  under 
the  skin.  For  example  ragweed  is  the  most  common  cause 
of  hay  fever.  Solutions  of  its  pollen,  made  in  standard  dilu- 
tions of  1-10,000  to  1-500  are  injected  in  rising  quantities, 
beginning  with  the  weakest.  Treatment  should  be  given  during 
the  winter  and  spring  so  that  as  high  a  degree  of  disensiti- 
zation  as  possible  will  be  accomplished.  Little  can  be  expected 
in  the  hay  fever  season. 


FIG.  19. — Illustrating    the    conception    of    deviation    of    complement. 
Amboceptor;  b,  antigen;  k,  complement.     (MacNeal.) 


.  Complement  Fixation. — Hemolysis  occurs  when  the  serum  of  a 
rabbit  immunized  against  washed  sheep's  red  blood  cells  is  mixed 
with  fresh  washed  sheep's  corpuscles  in  the  presence  of  comple- 
ment. If,  however,  complement  be  absorbed  in  any  way,  a  solu- 
tion of  the  coloring  matter  of  the  red  cells  will  not  occur  in  this 
mixture.  Complement  will  combine  with  anti-body  in  the  pres- 
ence of  antigen.  This  fact  has  been  taken  advantage  of  in  deter- 
mining both  the  nature  of  antigen  and  the  presence  of  anti-body. 
Its  most  important  practical  use  is  in  syphilis,  to  the  diagnosis  of 
which  Wassermann  applied  it,  and  the  Wassermann  test  is  for  the 
presence  of  syphilitic  anti-body  in  the  blood  serum  of  syphilitics. 


68  IMMUNITY 

This  test  is  positive  from  the  initial  lesions  all  during  life  unless 
the  patient  has  been  successfully  treated.  Indeed  the  para- 
syphilitic  states  also  give  it.  The  principles  of  the  test  are  also 
used  for  determining  the  presence  of  tuberculous,  leprous,  ty- 
phoid and  other  anti-bodies. 

The  materials  necessary  in  the  Wassermann  test  are  as  follows: 

i .  Syphilitic  antigen,  extract  from  the  syphilitic  liver  of  a  foetus, 
in  alcohol,  ether  or  water;  lipoids  like  lecithin  or  extracts  from 
guinea  pig's  heart  will  act  as  antigen. 

2  a.  Serum  from  a  known  case  of  syphilis  and  containing  there- 
fore syphilitic  anti-body. 

2b.  Known  non-syphilitic  serum  without  anti-body. 

3.  The  suspected  serum. 

4.  Fresh  serum  from  a  guinea  pig,  rich  in  complement. 

5.  Serum  from  a  rabbit  that  has  been  immunized  against 
washed  red  cells  from  a  sheep;  called  amboceptor. 

6.  Fresh  sheep's  red  blood  cells,  washed  in  saline  and  made  into 
5  percent  supension. 

The  solutions  are  all  standardized  so  that  only  sufficient  of  each 
is  added  to  complete  the  absorption  or  produce  the  hemolysis. 
The  serum  known  to  be  syphilitic  and  the  suspected  serum  are 
heated  to  56°C.  for  thirty  minutes  to  destroy  the  native  and  in- 
herent complement.  The  rabbit  anti-sheep  cell  serum  is  also 
heated  to  this  degree. 

The  hemolytic  series,  i.e.,  sheep's  cells,  rabbit's  anti-sheep's 
cells  serum  and  complement  are  standardized  to  find  out  what 
quantities  will  exactly  complete  hemolysis.  These  quantities 
are  the  units.  It  is  necessary  to  control  tests  to  find  out  what 
quantity  of  the  antigen  and  known  syphilitic  anti-body  will  unite 
to  bind  the  determined  quantity  of  complement.  The  tests  are 
performed  in  small  tubes  so  as  to  have  a  long  column  of  fluid  easier  1 
to  observe.  Tubes  are  set  as  follows: 


A. 


unit  #i     +i  unit  #2a  -f-  I  unit 
unit  #i     +i  unit  #3+1  unit 


unit  #1+1  unit  #20  +  I  unit  #4. 
unit  #2a  +  i  unit  #4. 
unit  #ab  +  i  unit  #4. 
unit  #3+i  unit  #4. 
unit  #4. 
unit  #i. 
unit  #2a. 
unit  #2b. 
unit  #3. 


FACTION                                          69 

unit  #5  + 

unit  #6  =  No  hemolysis. 

unit  #5  + 

unit  #6  = 

if  #3  be 

syphilitic,  no  hemolysis. 

if  #3  be 

^on-syphilitic  hemolysis. 

unit  #5  + 

unit  #6  =  Hemolysis. 

unit  #5  + 

unit  jj'6  =  Hemolysis. 

unit  #5  + 

unit  #6  =  Hemolysis. 

unit  #5  + 

unit  #6  =  Hemolysis. 

unit  #5  + 

unit  #6  =  Hemolysis. 

unit  #5  + 

unit  #6  =  No  hemolysis. 

unit  #5  + 

unit  #6  =  No  hemolysis. 

unit  #5  + 

unit  #6  =  No  hemolysis. 

unit  #5  + 

unit  #6  =  No  hemolysis. 

The  tubes  receive  first  the  solutions  on  the  left  and  are  placed 
in  the  37°C.  incubator  for  two  hours  to  allow  union  of  their  various 
parts,  particularly  the  complement  with  others.  They  then  re- 
ceive the  solutions  on  the  right,  are  placed  in  the  incubator  for 
half  an  hour  and  in  the  ice-box  overnight,  when  they  are  ex- 
amined for  a  solution  of  the  red  coloring  matter.  If  it  occurs,  the 
column  is  perfectly  clear  red  with  some  residue  of  extracted  cells. 
If  no  hemolysis  has  occurred,  the  red  cells  form  a  layer  at  the  bot- 
tom, and  the  column  is  clear  and  colorless. 

A  and  B  are  the  tests  of  syphilitic  sera  while  the  remaining  are 
to  find  out  if  the  other  solutions  affect  the  results  of  A  and  B.  Of 
course  tube  G  represents  simply  the  complete  hemolytic  system. 
The  extra  tests  are  to  exclude  the  possibility  of  interference  on 
the  part  of  any  single  member  with  the  complement  No.  4.  The 
character  of  the  test  is  found  in  tube  A  where  syphilitic  antigen  and 
serum  have  bound  or  fixed  the  complement  so  that  it  cannot  unite  with 
the  rabbit  serum  and  sheep's  corpuscles  to  hemolyze  the  latter.  This 
is  a  positive  test.  A  negative  test  is  when  hemolysis  occurs,  since  no 
anti-body  is  present  to  unite  with  complement  in  the  presence  of 
antigen. 

Complement  Deviation. — This  is  a  condition  arising  when  there 
is  too  much  amboceptor  and  too  little  complement.  The  free 
amboceptors  adsorb  complement  and  there  is  none  left  for  cell 
needs  or  renewed  demands.  It  is  to  be  distinguished  from  com- 
plement fixation.  The  terms  are  not  interchangeable. 


70  IMMUNITY 

ANTI-TOXINS,  VACCINES,  AND  TOXINS 

The  following  is  Wassermann's  list  of  anti-toxins: 
Anti-toxins  for  bacterial  toxins : 

Diphtheria 
Tetanus 
Botulism 
Pyocyaneus 
Symptomatic  Anthrax 

Anti-leucocidin,  an  anti-toxin  against  the  leucolytic  poinson  of 
staphylococcus 

Anti-toxins  for  the  blood  dissolving  toxins  of  certain  bacteria. 
Anti-toxin  for  animal  toxins : 

Anti-venene  for  snake  venom 

Anti-toxin  for  spider  poison 

Anti-toxin  for  scorpion  poison 

Anti-toxins  for  certain  poisons  in  fish,  eel,  salamander,  turtle,  and  wasp 

sera. 

Anti-toxins  for  plant  poisons : 
Anti-ricin  for  castor-oil  poison 
Anti-abrinforjequerity  bean  poison 
Anti-robin  for  locust  bean  poison 
Anti-croton  for  crotin-oil  bean  poison 
Anti-pollen  for  pollen  of  plants  that  produce  hay-fever. 

There  may  be  added  to  this  list  a  number  of  anti-sera  developed 
to  a  practical  value  in  recent  years  whose  activity  is  dependent 
not  on  anti-toxin  content,  but  upon  their  ability  to  agglutinate, 
precipitate,  and  opsonify  their  respective  microorganisms.  These 
sera  are  made  against  infections  with: 

Pneumococcus 

Meningitis  coccus 

Streptococcus 

Plague 

Dysentery  (bacillary) 

Staphylococcus 

Typhoid  fever 


MANUFACTURE   OF  ANTI-TOXINS  71 

The  first  two  have  proven  of  more  definite  value  than  the  rest. 

Manufacture  of  Anti-toxins. — If  small  doses  of  a  given  poison, 
such  as  diphtheria  toxin,  be  repeatedly  injected  into  a  susceptible 
animal,  and  if  the  dose  is  gradually  increased,  there  appears,  after 
a  time,  in  the  blood  serum,  an  anti-body,  or  anti-toxin.  This 
substance  in  the  serum  is  secreted  by  the  cells  and  corresponds  to 
the  free  receptors  in  Ehrlich's  lateral-chain  theory.  If  an  animal 
be  injected  with  the  anti- toxin,  and  then  with  a  large  dose  of  toxin 
— say  ten  times  the  amount  necessary  to  kill  it  if  it  had  not 
received  the  anti-toxin — it  will  not  be  harmed.  Here  the  free 
receptors  artifically  supplied  to  the  animal  unite  with  the  hapto- 
phorous  chains  in  the  toxin  molecule,  and  naturalize,  or  bind,  the 
toxophorous  or  poisonous  chains  in  the  molecule,  and  prevent 
toxophore  from  attacking  important  vital  cells  belonging  to  the 
animal.  And  if  the  anti- toxin  and  toxin,  after  being  mixed  in  a 
test-tube,  are  injected  into  a  susceptible  animal,  no  harm  results, 
if  they  are  in  proper  proportions,  since  the  same  thing  has  hap- 
pened in  vitro  that  happened  in  the  animal,  the  receptors  and 
haptophores  have  united;  the  toxophores  are  bound,  and  the 
animal  is  unharmed. 

The  manner  of  making  the  diphtheria  anti-toxin  can  be  taken 
as  a  type. 

Diphtheria  bacilli  are  grown  for  seven  to  ten  days  in  .  i  percent 
dextrose  bouillon  at  37°C.;  as  the  bacilli  grow  they  elaborate  a 
very  powerful  poison  or  toxin,  which  is  highly  complex  in  composi- 
tion. Strains  that  habitually  grow  on  the  surface  of  the  bouillon 
are  used,  the  access  of  air  enhancing  the  production  of  toxin.  It 
is  easily  decomposed  by  heat,  light  and  oxygen,  and  should  be 
used  soon  after  it  is  prepared.  After  the  cultures  have  grown  for 
several  days,  the  bouillon  is  filtered  through  a  porcelain  filter,  and 
is  then  stored  in  sterile  bottles  in  an  ice  chest.  Horses  are  gen- 
erally immunized,  since  they  are  susceptible  to  the  action  of  the 
toxin,  and  are  easily  managed.  Before  being  used  they  are  care- 
fully tested  with  tuberculin  for  tuberculosis  and  with  mallein  for 


72  IMMUNITY 

glanders.  Being  very  susceptible  to  infection  with  tetanus  while 
undergoing  treatment,  a  prophylactic  injection  of  tetanus  anti- 
toxin is  given  each  animal.  McFarland  found  that  the  death 
rate  from  tetanus,  in  a  large  stable,  was  greatly  reduced  after 
using  tetanus  anti-toxin  as  a  prophylactic  measure. 

Immunization  is  started  by  injecting  into  a  previously  examined 
healthy  horse  a  mixture  of  toxin  and  an ti- toxin  in  which  the 
former  is  not  quite  neutralized  by  the  latter.  These  quantities  are 
determined  by  guinea-pig  .tests.  Such  a  mixture  is  safer  for  the 
horse  and  begins  the  immunity  reaction  more  promptly.  A 
few  doses  like  this  are  given  after  which  pure  toxin  is  used. 
This  is  followed  by  a  rise  of  temperature,  local  reaction  and 
systemic  disturbance.  After  waiting  for  all  reactions  to  disappear 
injections  are  continued  by  slow  increases,  until,  after  a  few 
weeks  or  months,  1,000  c.c.  of  toxin  are  injected  at  one  time 
(enough  to  have  killed  a  dozen  horses  that  had  not  received  the 
smaller  doses  previously).  The  injection  of  the  toxin  is  followed 
by  an  immediate  fall  in  the  anti-toxic  power  of  the  serum,  only 
to  be  followed  by  a  quick  rise.  The  horse  will  not  produce  anti- 
toxin indefinitely.  After  the  animal  has  been  immunized  suffi- 
ciently, his  blood  is  drawn  from  the  jugular  vein,  and  after  the 
clot  has  formed  the  serum  is  drawn  off  and  stored. 

Anti-toxins  are  found  to  lie  in  the  pseudoglobulin  fraction  of 
the  serum.  Even  though  an  anti-serum  be  strong,  a  large  quan- 
tity would  have  to  be  injected  to  obtain  high  unit  value.  The 
globulins  of  the  whole  blood  are  now  precipitated  by  30  percent 
ammonium  sulphate  and  the  pseudoglobulins  are  dissolved  by 
normal  salt  solution.  This  concentrates  high  unit  values  into 
small  bulk.  When  removed  from  the  horse,  serum  may  contain 
300-800  units  per  cubic  centimeter.  After  concentration  a  value 
of  3-10,000  units  may  be  obtained. 

McFarland  found  that  a  horse  was  capable  of  producing  enough 
an  ti- toxin  to  protect  806  other  horses  against  doses  of  toxin,  each 
one  of  which  was  equivalent  to  the  total  amount  of  toxin  that  the 


ANTI-TOXINS  73 

immunized  horse  received.     Thus  there  is  evidently  a  tremendous 
overproduction  of  anti-toxin  far  above  the  needs  of  the  animal. 

The  various  component  parts  of  the  toxin  stimulate  the  cells  of 
the  horse  to  produce  the  receptors,  or  anti-toxin.  The  toxoids, 
themselves  not  poisonous,  have  the  property  of  stimulating  the 
production  of  anti-toxin.  We  measure  the  anti-toxic  powers  of 
the  anti- toxin  with  units  arbitrarily  devised.  An  anti-toxic  unit 
is  that  amount  of  horse  serum  just  necessary  to  protect  a  2$o-gram 
guinea  pig  against  100  times  the  minimum  lethal  dose  of  toxin. 

To  standardize  anti- toxin,  we  must  employ  animals,  into  the 
bodies  of  which  toxins  and  anti-toxins  are  injected.  If  a  certain 
amount  of  anti-toxin  is  necessary  to  protect  a  guinea  pig  against 
ten  times  the  minimum  fatal  dose  of  toxin  per  100  grams  of  guinea- 
pig  weight,  then  we  know  that  the  anti-toxin  contains  so  many 
units.  The  minimum  lethal  dose  of  toxin  is  the  smallest  quantity 
that  will  kill  a  guinea  pig  of  250  grams  in  four  days. 

A  standard  anti-toxin  is  kept  by  governments  to  be  used  as  a 
control  of  the  products  of  biological  chemists. 

Against  this  standard  anti-toxin  a  toxin  of  unknown  strength  is 
measured  by  means  of  guinea  pigs.  The  toxin  unit  thus  found  is 
then  used  to  determine  the  anti-toxic  unit  of  anti-toxins  of  un- 
known power. 

Anti- toxic  serum  is  preserved  by  the  addition  of  .5  percent  of 
tri-cresol  or  phenol.  It  remains  practically  unchanged  in  strength 
for  a  year  or  more. 

It  is  not  only  of  value  as  a  curative  agent,  neutralizing  the  toxins 
already  formed,  but  is  valuable  as  an  immunizing  one  against  in- 
fection. Persons  exposed  to  diphtheria  and  giving  a  positive 
Schick  test  should  be  given  an  immunizing  dose  of  1,000  to  1,500 
units.  If  injected  early  in  a  case  of  diphtheria,  it  is  much  more 
likely  to  do  good,  than  if  used  later.  Some  desperate  cases  have 
received  100,000  units  and  have  recovered.  The  following  is  the 
very  good  guide  given  by  Park: 


74  IMMUNITY 

SINGLE  DOSE  ONLY 
Infant,  10  to  30  pounds  (under  2  years) 

Mild  Moderate  Severe  Malignant 

2,000  3,000  5,000 

3,000  5,ooo  10,000  10,000 

Child,  30  to  90  pounds  (under  15  years) 

3,000  4,000  10,000  10,000 

4,000  10,000  15,000  20,000 

Adults,  90  pounds  and  over 

3,000  S,ooo  10,000  15,000 

5,000  10,000  20,000  40,000 

Method  of  Administration 

Intramuscular 


or 

^  Intravenous 

Subcutaneous 

Intramuscular 

%  Intravenous 

and 

or 

or 

and 

3^  Intramuscular 

Intramuscular 

Subcutaneous 

%  Intramuscular 

or 

or 

Subcutaneous 

Subcutaneous 

Tetanus  Anti-toxin. — Tetanus  anti-toxin  is  produced  in  a  man- 
ner similar  to  that  of  diphtheria  anti-toxin.  As  the  horse  is  ex- 
ceedingly sensitive  to  tetanus  toxin,  before  the  immunizing  process 
is  begun,  the  toxin  is  attenuated  by  heat  or  iodine. 

The  an ti- toxin  is  standardized,  as  in  diphtheria,  by  testing  its 
potency  against  the  toxin.  A  guinea  pig  of  500  grams  weight  is 
used,  and  test  toxin  is  employed  of  such  strength  that  .01  c.c.  will 
kill  the  guinea  pig  in  about  four  days.  The  United  States  unit 
of  tetanus  an  ti- toxin  is  now  the  least  quantity  of  an  ti- tetanic 
serum  necessary  to  save  the  life  of  a  350-gram  guinea  pig  for 
ninety-six  hours  against  the  official  test  dose  of  standard  toxin 
furnished  by  the  Hygienic  Laboratory  of  the  Public  Health 
Service. 

The  toxin  of  the  tetanus  bacillus  has  such  an  affinity  for  nervous 
tissue  that,  once  it  becomes  attached,  separation  is  difficult,  or 
even  impossible  if  union  has  existed  for  some  time.  More  than 
this  the  toxin  has  a  greater  affinity  for  nervous  tissue  than  for  its 


ANTI-TOXINS  75 

own  anti-toxin.  If  an  animal  be  injected  intravenously  with 
anti-toxin  and  intracerebrally  with  toxin  it  will  die  of  tetanus. 
This  explains  why  the  use  of  anti-toxin  in  the  treatment  of  the 
diesase  must  be  vigorous,  early  and  direct.  Dosages  of  10-50,000 
units  are  necessary  before  symptoms  have  become  well  estab- 
lished. Subcutaneous  methods  are  too  slow  and  indirect.  Anti- 
toxin should  be  given  into  the  spinal  canal,  into  the  vein  and 
around  the  wound.  Prophylactically  anti-serum  should  be  given 
in  every  case  of  penetrating,  lacerated  wound  especially  if  soiled 
with  earth,  street  dirt,  rust  or  gun  waddings.  Tetanus  antitoxin 
is  more  efficient  as  a  prophylactic  than  as  a  remedy. 

Streptococcus  Antiserum. — The  three  principal  groups  of 
streptococci,  hemolytic,  non-hemolytic  and  viridans,  have  differ- 
ent immunity  factors  and  each  kind  produces  it  own  anti-bodies. 
These  however  are  never  in  great  amount  and  this  seems 
due  to  the  rather  feeble  antigenic  power  of  the  cocci  them- 
selves. The  only  useful  anti-sera  are  those  prepared  against  a 
large  number  of  strains  of  each  of  the  varieties  by  repeated 
injections  over  a  long  period.  Their  antigenic  fractions  seem  to 
be  hemolytic,  leucocytolytic  and  neurolytic.  The  anti-bodies 
formed  in  horse's  serum  seem  to  decrease  in  value  after  removal 
from  the  body.  They  are  chiefly  agglutinative  and  anti-toxic. 
For  clinical  use  the  serum  should  be  as  fresh  as  possible  and  used 
intravenously  in  doses  of  50  to  200  c.c.  It  has  been  employed 
in  endocarditis,  osteomyelitic  and  puerperal  septicemia  with  some 
promise. 

The  Anti-pneumococcus  serum  is  prepared  in  the  same  way. 
Horses  are  immunized  by  the  injection  of  first  autolysates  then 
living  cultures,  and  the  horse's  blood,  after  a  period  of  treatment 
by  cultures,  is  drawn  off,  preserved  with  tri-cresol.  It  is  used  in 
the  crude  form  of  serum  as  the  anti-bacterial  powers  are  not  easily 
concentrated.  It  is  standardized  so  that  .2  c.c.  shall  protect  a 
mouse  against  100,000  times  the  amount  of  a  culture  of  pneumo- 
cocci  that  would  kill  a  control  mouse.  It  has  been  found  that 


76  IMMUNITY 

there  are  in  this  country  four  groups  of  pneumococci  in  a  serolog- 
ical  sense,  a  discovery  confirmed  by  therapeutic  results.  It  is 
possible  to  prepare  curative  anti-sera  against  two  of  the  groups 
while  the  other  two  fail  to  call  forth  useful  anti-bodies.  In  any 
given  case  of  pneumonia  the  type  of  infecting  organism  is  deter- 
mined by  isolating  it  from  the  sputum  and  performing  the  agglu- 
tination test  with  the  individual  sera  of  Types  I  and  II,  whereupon 
should  one  of  these  react  the  appropriate  anti-serum  can  be  in- 
jected. The  organisms  are  obtained  for  the  agglutination  test 
by  injecting  the  sputum  into  a  mouse's  peritoneum,  killing  the 
animal  after  six  to  eight  hours  and  using  the  rich  growth  of  cocci 
in  the  peritoneal  fluid  as  the  bacterial  suspension.  With  Type  I 
the  therapeutic  results  are  very  promising;  with  II  helpful  at  times. 
Type  III  is  the  Pneumococcus  mucosus  yielding  no  useful  anti- 
serum,  while  Type  IV  is  a  heterogeneous  group  possessing  no 
serological  uniformity  and  unable  to  call  forth  any  valuable  anti- 
body in  the  injected  horse.  The  serum  is  given  intravenously  in 
dose  of  50-100  c.c.  and  repeated  when  the  temperature  rises 
again,  sometimes  every  eight  hours. 

Meningitis  Anti-serum. — Epidemic  meningitis,  caused  by  the 
Micrococcus  meningitidis  intracellularis  or  meningococcus,  can 
be  treated  by  anti-serum.  The  cocci  owe  their  power  to  endo- 
toxins  and  pus  formation,  exerted  chiefly  in  the  coverings  of  the 
central  nervous  system.  They  are  present  in  the  spinal  fluid, 
only  being  found  in  the  blood  early  in  an  ordinary  attack  or 
in  highly  septicemic  cases.  Anti-serum  is  made  by  injecting 
horses  with  first  dead  then  living  bacteria  until  its  serum  shall 
have  acquired  high  agglutinative,  opsonic  and  bactericidal 
power.  The  horse's  serum  is  separated,  preserved  as  usual,  and  - 
used  by  injecting  into  the  arachnoid  space  by  lumbar  or  cranial 
puncture.  This  route  is  selected  to  bring  the  anti-serum  into 
close  relation  with  the  cocci  since  by  the  subcutaneous  or  intra-  * 
venous  routes  insufficient  anti-bodies  pass  from  the  blood  to  the 
meningeal  fluid.  It  should  be  given  also  into  the  blood  stream 


VACCINATION  77 

in  order  to  prevent  septicemia.  Its  effect  is  to  increase  phagocy- 
tosis and  cause  bacteriolysis.  No  satisfactory  standard  has  been 
devised.  Dosage  varies  but  a  safe  guide  is  to  inject  under  the 
meninges  75  percent  as  much  as  the  fluid  removed  at  lumbar 
puncture.  Intravenous  dosage  should  be  20-50  c.c.  The  anti- 
serum  probably  has  no  prophylactic  value. 

Anti-plague  Serum. — Yersin,  a  French  bacteriologist,  treated 
horses  with  living  cultures  of  plague  bacilli,  and  after  a  long  period 
of  immunization  used  a  serum  which  either  effectually  vaccinated 
an  individual  against  the  plague,  or  greatly  modified  the  disease 
after  it  had  once  begun. 

The  action  of  the  serum  is  bactericidal,  as  well  as  anti-toxic. 
The  dose  varies  with  the  stage  of  the  disease;  20  c.c.  is  an  effective 
prophylactic  dose,  while  from  20  to  300  c.c.  have  been  used  often 
as  curative  doses. 

VACCINATION 

By  the  use  of  attenuated,  or  killed  microorganisms,  it  is  possible 
to  effectively  vaccinate  men  and  animals  against  many  diseases, 
notably,  small-pox,  hydrophobia,  plague,  cholera,  typhoid  fever, 
anthrax  and  quarter-evil. 

Any  of  the  bacterial  products  used  as  prophylactics  are  some- 
times called  vaccines,  the  word  being  borrowed  from  small-pox 
vaccine.  It  is  better  to  use  the  word  bacterin  for  the  purpose, 
even  when  they  are  given  prophylactically.  Bacterin  is  employed 
for  the  dead  bacterial  masses  used  therapeutically.  Sensitized 
bacterins  are  vaccines  that  have  been  exposed  to  the  action  of  the 
respective  anti-sera  before  their  use.  It  is  the  purpose  of  this 
procedure  to  prepare  the  organisms  by  combining  them  with  the 
homologous  anti-bodies  so  that  when  injected  they  have  only  to 
combine  with  complement  to  begin  their  immunizing  effect. 
Theoretically  this  is  correct  but  the  practical  value  has  not  been 
fully  confirmed. 


78  IMMUNITY 

Vaccination  Against  Small-pox 

Jenner's  observations  in  1794  established  the  etiological  rela- 
tionship between  human  variola  and  cow-pox.  Human  small- 
pox virus  passed  through  a  cow  will  lose  its  power  to  produce 
typical  variola  but  will  produce  in  man  a  local  condition  called 
vaccinia  which,  upon  recovery,  leaves  behind  immunity  to 
variola. 

By  the  term  vaccination,  in  its  strict  sense,  we  mean  the  applica- 
tion of  attenuated  small-pox  virus,  weakened  by  passage  through 
kine,  to  human  beings  and  infecting  them  with  the  modified 
disease.  The  disease  is  localized  at  first  at  the  site  of  inoculation, 
and  a  bleb  or  vesicle  forms.  As  a  rule  the  disease  does  not  become 
generalized.  It  creates,  in  the  vaccinated  individual,  an  active 
immunity  against  small-pox.  The  toxins  diffused  through  the 
blood-stream  stimulate  the  cells  of  the  body  into  forming  either 
anti-toxic  or  other  anti-bodies.  These  various  substances,  as 
yet  unknown,  remain  for  a  long  period  within  the  body  of  the 
vaccinated  person  and  may  protect  it  for  years  against  invasion 
and  infection  with  the  cytorcytes  in  virulent  form.  A  person  who 
has  variola  cannot  be  vaccinated,  subsequently  he  is  immunized 
against  vaccinia  by  this  attack  of  variola,  just  as  he  can^be  immun- 
ized against  variola  by  vaccinia  infection. 

Since  Jenner  first  discovered  that  cow-pox  introduced  into  the 
body  prevented  small-pox,  it  has  been  the  world- wide  custom  to 
use  either  the  dried  virus  or  liquid  glycerinized  virus  from  the  cow 
or  human  beings  in  the  process  of  vaccination.  It  has  been  found 
that  human  virus  generally  used  was  likely  in  rare  instances  to 
transmit  syphilis,  so  it  is  now  the  universal  custom  to  use  cow 
virus.  This  virus  is  collected  from  fresh  vesicles  in  calves  or 
young  heifers,  as  clean  as  possible,  as  it  is  used  as  seed  to  inoculate 
the  animals  and  the  operation  is  done  under  strict  antiseptic 
precaution.  After  a  week  the  virus  is  collected  under  similar 
antiseptic  precautions  by  scraping  the  base  of  the  vesicle  with  a 


VACCINATION  AGAINST   CHOLERA  79 

sterile  curette.  The  pulpy  substance  thus  obtained  is  mixed 
with  glycerine  and  stored  for  a  month  or  more.  The  action  of  the 
glycerine  is  to  rid  the  virus  of  many  of  the  bacteria,  through,  it  is 
supposed,  a  hydroly  tic  action.  This  virus  is  then  rubbed  into  the 
skin  of  the  individual  to  be  vaccinated  under  strict  aseptic  pre- 
cautions. At  the  end  of  a  week,  a  pearly  white  vesicle  is  formed, 
and  it  is  then  considered  that  vaccination  has  "taken"  and  that 
the  individual  is  protected  against  variola.  This  action  of 
immunization  is  supposed  to  be  complete  on  the  fourth  day  after 
the  virus  has  been  introduced.  This  is  a  matter  that  is  difficult 
to  decide,  but  the  immunization  process  is,  no  doubt,  a  very  slow 
one,  like  every  other  immunizing  process  where  the  immunity  is 
autogenous  and  active,  and  not  passive,  as  in  the  case  of  diph- 
theria anti-toxin. 

Persons  who  are  entirely  immune  to  variola  will  show  twenty- 
four  hours  after  vaccination  a  distinct  red  areola  without  vesicles 
or  pustules;  this  is  the  reaction  of  immunity.  Recent  successful 
vaccination  leaves  the  patient  in  a  condition  of  resistance  and 
four  to  six  days  after  revaccination  an  areola  surmounted  by  a 
reddish  papule  is  seen;  this  is  vaccinoid. 

Vaccination  Against  Cholera 

By  the  attenuations  of  cholera  spirilla,  Haffkine  has  produced 
vaccines  which  effectively  protect  individuals  against  infection 
with  cholera,  or  if  they  become  infected  with  the  disease,  it  is  so 
modified  that  they  can,  and  do,  more  easily  recover.  He  employs 
two  vaccines,  a  weak  one  and  a  stronger  one.  The  weak  one  is 
used  to  prepare  for  the  stronger  one,  which  is  the  effective  vaccine. 

The  weak,  or  first  virus,  is  prepared  by  growing  the  cholera 
vibrios  at  a  high  temperature,  39°C.,  in  a  current  of  air.  The 
stronger  is  prepared  by  passing  the  vibrios  through  a  series  of 
guinea  pigs,  so  increasing  the  virulence  that  the  virus  is  invariably 
fatal  to  the  guinea  pigs  in  eight  hours.  After  cultivating  this 


8o 


IMMUNITY 


virus  on  agar,  the  surface  growth  is  washed  off  with  sterile  water 
(8  c.c.)  and  %  part  of  this  is  used  as  a  dose.  As  the  virus  rapidly 
attenuates  it  must  be  reactivated  by  passing  it  through  guinea 
pigs  from  time  to  time. 

The  first  injection  is  given  in  the  flank,  and  the  second  follows 
in  five  days.  Accordingly  as  the  symptoms  are  severe,  so  will  the 
resulting  protection  be  strong.  Haffkine  has  given  70,000  injec- 
tions without  an  accident.  The  following  results  were  obtained 
by  Haffkine  who  worked  in  India  for  the  British  Government: 


Population 

Case  —  Cholera 

Deaths 

Total 

Percent 

Total 

Percent 

Non-inoculated,  1,735  

171 

21 

10.63 
4.20 

ii.  3 
19.0 

6.51 
3-80 

Inoculated,  500            

The  immunity  conferred  by  this  mode  of  vaccination  is  not 
complete  until  ten  days  after  treatment.  It  is  possible  to  vac- 
cinate with  these  relatively  virulent  bacteria  because  they  are 
given  under  the  skin,  a  place  where  life  of  the  vibrios  soon  ceases. 

During  an  attack  of  cholera  the  vibrios  do  not  enter  the  blood 
but  remain  in  the  deep  layers  of  the  intestinal  mucosa. 

Vaccination  Against  Typhoid 

By  the  injection  of  sterilized  cultures  of  typhoid  bacilli,  it  is 
possible  to  create  an  immunity  of  a  moderate  kind  against  enteric 
fever.  The  method  was  perfected  by  Wright,  and  his  mode 
of  procedure  is  to  secure  a  culture  of  typhoid  both  virulent  and 
able  to  call  forth  a  large  amount  of  anti-body  in  the  injected 
person,  which  is  tested  on  guinea  pigs,  and  the  minimum  lethal 
dose  for  a  loo-gram  guinea  pig  is  used  as  the  dose  for  man.  This 
dose  varies  from  .5  c.c.  to  1.5  c.c.  of  a  culture  sterilized  by  heat 


ANTI-TYPHOID  VACCINATION  8 1 

at  6o°C.,  and  preserved  with  lysol.  After  the  injection  there  is 
often  redness  and  pain  at  the  site  of  inoculation,  some  fever  and 
lymphangitis.  The  method  at  present  in  use  in  the  United 
States  is  to  employ  a  twenty-four  hour  agar  slant  or  bouillon 
culture,  killed  by  heating  as  above;  these  are  suspended  in 
saline  with  trikresol  or  phenol  and  counted  in  a  hemocytometer. 
In  order  to  control  the  toxicity  of  this  suspension  a  mouse  is  in- 
jected subcutaneously  with  a  billion;  it  should  live  at  least  five 
days.  The  doses  are  500  million,  1,000  million  and  1,000  million 
eight  to  ten  days  apart. 

The  results  of  Col.  F.  F.  Russell,  U.  S.  A.,  a  man  who  has  had 
much  experience,  since  he  was  in  charge  of  the  army  vaccinations, 
are  interesting  and  instructive.  He  says : 

1.  "  Anti- typhoid  vaccinations  in  healthy  persons  is  a  harmless 
procedure. 

2.  It  confers  almost  absolute  immunity  against  infection. 

3.  It  is  the  principal  cause  of  the  immunity  of  our  troops 
against  typhoid  in  the  recent  Texas  maneuvers. 

4.  The  duration  of  the  immunity  is  not  yet  determined,  but 
is  assuredly  two  and  one-half  years  and  probably  longer. 

5.  Only  in  exceptional  cases  does  its  administration  cause  an 
appreciable  degree  of  personal  discomfort. 

6.  It  apparently  protects  against  the  chronic  bacillus  carriers, 
and  is  at  present  the  only  means  by  which  a  person  can  be  pro- 
tected against  typhoid  under  all  conditions. 

7.  All  persons  whose  profession  or  duty  involves  contact  with 
the  sick  should  be  immunized. 

8.  The  general  vaccination  of  an  entire  community  is  feasible 
and  could  be  done  without  interfering  with  general  sanitary 
improvements  and  should  be  urged  wherever  the  typhoid  rate 
is  high." 

The  wisdom  of  these  conclusions  has  been  abundantly  proven 
in  our  army  during  the  World  War  when  practically  no  typhoid 
fever  occurred  in  vaccinated  men.  It  might  be  added  that  no 


82  IMMUNITY 

immunizing  procedure  is  perfect  and  this  one  does  not  justify 
the  drinking  of  water  known  to  be  polluted. 

Vaccination  against  all  the  typhoid  fevers,  due  to  the  typhoid 
bacillus,  the  paratyphoid  bacillus  A  and  B,  can  be  accomplished 
by  combining  these  organisms  in  one  vaccine.  The  form  now  in 
common  use,  as  employed  by  our  armed  forces,  is  one  suspension 
in  each  cubic  centimeter  of  which  are  contained  1,000  million 
typhoid  bacillus,  750  million  of  paratyphoid  A  and  750  million 
of  paratyphoid  B.  Three  doses,  .5  c.c.,  i  c.c.  and  i  c.c  are  given 
ten  days  apart.  This  makes  a  total  of  6,250  million  organisms 
injected. 

Vaccination  Against  Pneumococcus  Infections 

Experiences  in  South  Africa  by  Lister  and  in  the  American 
Army  by  Cecil  seem  to  hold  out  encouragement  for  the  prophy- 
laxis of  pneumonia  by  vaccines  of  the  respective  coccus.  The 
types  of  pneumococci  prevalent  in  a  district  must  be  determined 
and  used  in  the  suspension.  The  preparations  employed  by 
Cecil  contained  the  three  fixed  American  types  and  were  given 
in  doses  of  6,000  million  four  times  at  six  to  eight  day  intervals. 
Under  war  conditions  some  advantage  was  observed  but  available 
data  are  too  few  to  form  a  general  evaluation.  Experimenta- 
tion and  human  experience  indicate  the  harmlessness  of  this 
procedure  and  use  is  recommended.  The  action  of  the  vaccine 
seems  to  be  stimulation  of  opsonins  and  of  bacteriolysins. 

Vaccination  Against  Diphtheria 

According  to  the  experiments  of  Behring,  Theobald  Smith  and 
W.  H.  Park  it  is  perfectly  feasible  and  without  danger  to  immun- 
ize children  against  diphtheria.  As  already  mentioned  immuni- 
zation of  horses  is  begun  with  nearly  neutral  toxin-anti-toxin 
mixtures.  For  immunization  of  children  only  such  are  used. 
Solutions  of  the  toxin  and  anti- toxin  are  made  to  contain  i  unit 


VACCINATION   AGAINST   ANTHRAX  83 

of  the  latter  and  60  percent  and  80  percent  of  a  unit  of  the  former. 
Two  or  three  injections  are  given,  the  first  or  the  first  and  second 
of  the  former  strength,  the  last  having  the  higher  value  of  toxin. 
A  local  reaction  occurs  and  not  infrequently  a  general  one — 
fever,  malaise.  Immunity  requires  a  week  to  start  and  about  a 
month  to  be  fully  developed.  It  is  believed  to  last  at  least 
four  years.  Immunization  need  only  be  given  to  those  who  have 
a  positive  Schick  test. 

Vaccination  Against  Plague 

Haffkine,  in  India,  has  vaccinated  natives  and  others  against 
plague  by  somewhat  the  same  methods  employed  in  anti-cholera 
vaccination.  The  B.  pestis  is  cultivated  in  flasks  of  bouillon;  as 
it  grows,  the  stalactite-like  scum  on  top  is  shaken  from  time  to 
time  to  the  bottom  of  the  flask.  After  growing  for  six  weeks  in 
the  bouillon,  the  culture  is  killed  at  7o°C.  for  three  hours.  It  is 
then  used  as  vaccine,  3  c.c.  is  the  usual  dose  for  man,  2  c.c.  for 
woman,  and  children  still  less.  After  the  inoculation,  heat  and 
redness  appear  at  the  site  of  inoculation,  and  the  patient  feels  ill 
and  has  some  fever.  Haffkine  holds  that  immunity  against  the 
plague  is  complete  in  twenty-four  hours  after  vaccination.  His 
results  are  at  times  really  very  good. 

The  Indian  Plague  Commission  reported  that  the  measure  was 
valuable  as  a  means  of  preventing  infection;  while  it  was  not  an 
absolutely  certain  'means,  yet  it  sensibly  diminished  the  death 
rate.  The  immunity  lasts  about  a  month.  Such  vaccines  are 
not  to  be  used  after  attack  has  started. 

Vaccination  Against  Anthrax 

Of  all  forms  of  vaccination  against  disease  with  attenuated 
bacteria  this  is  the  most  successful.  Its  use  is  confined  to  domes- 
tic animals,  sheep,  cattle,  and  horses,  and  has  reduced  the 
mortality  in  the  country  where  it  is  used  from  10  percent  to  .5 


84  IMMUNITY 

percent.  The  method  requires  the  employment  of  two  vac- 
cines made  of  attenuated  anthrax  bacilli.  No.  i  is  a  culture  of 
bacilli  attenuated  by  growing  them  at  a  high  temperature,  42.5°C., 
in  a  current  of  air  for  twenty-four  days.  No.  2  is  grown  at  the 
same  temperature  for  only  twelve  days.  The  first  vaccine  is 
used  to  immunize  the  animal  against  the  second,  which  causes  a 
marked  local  reaction,  and  which  is  the  real  immunization  agent 
against  infection  with  virulent  anthrax  bacilli.  The  injections 
are  given  about  one  week  apart.  Many  State  Governments  as 
well  as  the  Federal  Government  of  the  United  States  supply 
the  vaccine  gratis  to  stock  raisers  and  others. 

Vaccination  Against  Black-leg  or  Quarter-evil 

Quarter-evil,  or  Rauschbrarid,  is  due  to  a  specific  bacillus. 
Vaccination  against  this  disease  may  be  accomplished  by  inocu- 
lating with  a  powder  consisting  of  dried  muscle  from  the  affected 
part  of  infected  animal.  There  are  two  vaccines,  No.  i,  and  No. 
2.  The  first  is  prepared  by  heating  (and  thus  attenuating)  the 
bacilli  up  to  103 °C.  The  second  is  prepared  by  raising  the  tem- 
perature up  to  Q3°C.  These  vaccines  are  given  at  a  short  time 
apart,  and  the  immunity  is  effective.  The  method  is  valuable 
to  stockmen. 

Vaccination  Against  Tuberculosis 

There  is  at  present  no  safe  and  satisfactory  prophylactic  meas- 
ure for  the  production  of  increased  resistance  in  man  to  tubercu- 
losis. In  cattle  the  repeated  injection  of  bacilli  of  the  human  type 
has  been  found  capable  of  raising  the  animal's  resisting  power  to 
a  sort  of  immunity.  The  therapeutic  use  of  the  various  tubercu- 
lines  has  on  the  other  hand,  met  with  better  success,  and  it  seems 
that  for  human  medicine  at  least  that  they  have  won  a  place  in 
the  treatment  of  surgical  tuberculosis.  They  may  be  also  of 
value  in  pulmonary  disease. 


THE    TUBERCULINS  85 

The  Tuberculins 

The  toxin  of  the  tubercle  bacilli  (old  tuberculin)  is  prepared  by 
growing  the  organism  for  a  long  period  in  glycerinized  veal  broth, 
after  which  the  flasks  are  steamed  in  a  sterilizer  for  an  hour  or 
more,  and  then  the  bacilli  are  filtered  out  through  porcelain  filters. 
The  filtrate  is  reduced  by  boiling  to  one-tenth  of  its  bulk,  and  to 
this  .5  percent  of  carbolic  acid  is  added  as  a  preservative.  If 
this  toxin,  even  in  minute  doses,  is  injected  under  the  skin  of  a 
tuberculous  animal,  it  acts  as  a  powerful  poison.  In  a  few  hours, 
it  causes  a  rapid  rise  of  body  temperature,  accompanied  by  nausea 
and,  perhaps,  vomiting.  About  the  localized  foci  of  tuberculosis, 
a  vigorous  reaction  occurs.  Around  indolent  old  sores  and  other 
lesions  there  is  a  tendency  to  heal  by  the  casting  off  of  necrosed 
tissues,  and  the  infiltration  of  the  perituberculous  area  with  leu- 
cocytes. In  lupus  (tuberculosis  of  the  skin)  redness  and  heat 
occur  about  the  lesion.  This  febrile  phenomenon  following  the 
injection  of  tuberculin  into  tuberculous  animals  is  a  valuable 
diagnostic  feature  toward  the  recognition  of  tuberculosis  in 
animals  and  in  man.  In  90  percent  of  cases  the  reaction  is 
trustworthy. 

Its  use  in  man  has  been  much  questioned,  as  it  is  thought  by 
some  to  disseminate  the  disease  from  original  and  confined  foci. 
This,  however,  has  been  denied.  Many  able  clinicians  use  it  and 
recommend  it  (Osier,  Trudeau,  Musser). 

Koch's  new,  or  T.R.  tuberculin  was,  like  the  old,  designed  by 
him  as  a  therapeutic  agent  for  the  cure  of  tuberculosis.  It  is 
made  by  pulverizing  the  bodies  of  living  tubercle  bacilli  and  dis- 
solving the  residuum  in  an  indifferent  fluid,  centrifuging  this  and 
collecting  the  sediment  which  is  Tuberculin  Rest,  T.R.  The 
solution  above  this  sediment  containing  soluble  substances  from 
the  bacillary  bodies  is  Tuberculin  Obers,  T.O.  It  produces  a 
more  intense  reaction  than  the  old  tuberculin.  Like  the  old,  it 
is  used  in  the  treatment  of  lung,  bone,  laryngeal,  and  skin  tuber- 


86  IMMUNITY 

culosis.     It  certainly  causes  a  local  reaction  about  tuberculous 
foci,  and  no  doubt  aids  in  the  building  up  of  healthy  tissue. 

The  dose  of  tuberculin  for  testing  purposes  should  be  .5-2.  mg. 
for  a  child,  2-6.  mg.  for  a  young  or  weak  person,  and  5--io.  mg. 
for  a  larger  person.  It  is  well  to  give  the  highest  dose  that  it  is 
believed  the  patient  will  stand  in  order  to  get  a  prompt  and 
definite  result  thus  avoiding  the  necessity  of  a  repetition.  Re- 
peating the  injection  of  such  amounts  is  not  without  danger  as 
it  might  light  up  a  latent  lesion.  Any  focus  that  is  at  the  bottom 
of  a  clinical  condition  requiring  such  a  test,  will  give  a  positive 
reaction  with  the  quantities  mentioned.  For  therapeutic  pur- 
poses one  begins  with  an  injection  of  .000000 1  gram  or  smaller 
and  increases  .slowly  according  to  the  patient's  condition.  Tuber- 
culin should  only  be  administered  by  experts. 

Ma  lie  in 

Mallein  is  a  preparation  made  from  the  toxin  of  the  glanders 
bacilli,  and  is  prepared  precisely  as  the  old  tuberculin.  By  in- 
creasing the  virulence  of  the  glanders  bacilli,  by  passage  through 
a  series  of  guinea  pigs,  a  highly  virulent  bacillus  is  obtained.  It 
is  then  grown  in  glycerinized  bouillon  for  a  month  at  37°C.  The 
resulting  fluid  is  sterilized  by  heat  and  filtered  through  a  Pasteur 
filter.  The  filtrate  is  evaporated  to  one-tenth  its  quantity 
when  intended  for  conjunctival  use  or  left  in  its  natural  state 
when  for  subcutaneous  use.  A  small  amount  of  carbolic  acid 
is  added  in  order  to  preserve  it. 

In  a  horse  with  glanders,  the  injection  of  mallein  is  followed  by 
a  large  painful  swelling  at  the  injection  site.  With  this  there 
is  a  rise  of  temperature,  which  is  the  diagnostic  reaction  that 
indicates  infection  with  glanders.  In  this  respect  the  reaction 
is  like  tuberculin.  In  healthy  horses  no  rise  of  temperature 
follows  the  injection,  and  the  resulting  swelling  more  quickly  sub- 
sides. A  convenient  test  is  the  introduction  of  a  drop  of  concen- 
trated mallein  into  the  conjunctival  sac.  Positive  reaction  is 


IMMUNIZATION   AGAINST  HYDROPHOBIA  87 

indicated  by  lacrimation  and  purulent  collections  within  twenty- 
four  hours.  Mallein  has  been  used  as  a  prophylactic  agent 
against  glanders  with  some  success,  and  treatment  can  be  carried 
on  with  it  in  valuable  animals. 

Immunization  Against  Hydrophobia 

While  the  actual  causal  agent  of  hydrophobia  has  thus  far 
eluded  bacteriologists,  certain  well-marked  histologic  lesions  have 
been  discovered  in  the  ganglia  of  the  central  nervous  system,  and 
in  the  medulla,  which  are  not  found  in  any  other  disease.  This 
dispels  all  doubt  as  to  the  fact  that  hydrophobia  is  a  real  clinical 
entity. 

It  is  possible  to  immunize  animals  and  man  against  this  disease 
by  the  use  of  attenuated  virus.  In  common  with  many  other 
viruses,  that  of  hydrophobia  can  be  weakened  through  the  action 
of  either  heat,  drying,  light,  or  chemicals.  Pasteur  found  that  by 
drying  the  spinal  cords  of  rabid  animals  for  two  weeks,  they  be- 
come totally  avirulent.  If  the  cord  is  dried  but  three  or  four  days, 
the  virulence  is  but  slightly  reduced.  Immunity  to  rabies  can  be 
produced  by  injecting  minute  quantities  of  the  poison,  and  then 
gradually  increasing  the  dose  until  virulent  virus  can  be  employed. 

Recent  work  seems  to  indicate  that  simple  dilutions  of  the 
virus  so  that  minute  quantities  are  used,  can  be  employed 
prophylactically  instead  of  dried  material.  .  ' 

Modification  of  the  amount  of  poison  used  may  be  affected  by 
employing  equal  quantities  of  spinal  cords  from  rabid  animals  that 
have  dried  varying  lengths  of  time.  The  vaccine  consists  of  pieces 
of  cord,  i  cm.  in  length,  from  rabbits  that  have  been  killed  by 
inoculation  with  fixed  virus.  This  is  emulsified  with  sterile  salt 
solution.  Cord  that  has  dried  for  fourteen  days  is  first  injected, 
after  which  cords  that  have  dried  fewer  and  fewer  days,  until, 
finally,  one  that  has  dried  only  three  days  is  injected. 

In  cases  of  bites  by  rabid  dogs  on  the  face  or  head,  the  vaccina- 
tion must  be  rapid,  so  two  injections  per  diem  are  given.  In 


88  IMMUNITY 

Berlin  the  weakest  injection  used  (the  first)  is  made  from  a  cord 
that  has  dried  but  eight  days,  and  the  course  is  much  quicker. 
It  was  at  first  thought  that  short  drying  might  carry  over  too 
much  virus  but  in  order  to  treat  certain  serious  head  bites,  cords 
of  3  and  4  days  drying  were  tried  not  only  without  damage  but 
with  promising  results.  Now  in  threatening  cases  treatment 
may  be  begun  with  3  day  cords,  then  2  day  cords.  The  effect 
of  this  mode  of  inoculation  is  to  produce  in  the  bitten  individual 
a  very  rapid  active  immunity,  quicker  in  its  action  than  the 
infection.  The  treatment  is  solely  prophylactic  and  in  no  way 
curative.  If  symptoms  of  rabies  have  set  in,  the  treatment 
is  of  no  avail.  In  rabies  the  incubation  period  is  about  six  weeks, 
so  that  there  is  plenty  of  time  to  immunize  the  patient  by  injec- 
tion with  attenuated  virus. 

Since  the  immunizing  process  is  always  begun  after  the  bite  of  a 
rabid,  or  supposedly  rabid  dog,  it  differs  from  other  vaccinations, 
which  are  resorted  to  before  infection. 

Results  of  Treatment. — Among  those  bitten  by  rabid  animals 
the  total  mortality  before  the  introduction  of  vaccination  was  not 
less  than  10  percent.  Among  the  same  class  of  patients  in  the 
Pasteur  institutes,  the  death  rate  of  all  cases,  early  and  late,  has 
been  reduced  to  a  fraction  of  i  percent.  Those  cases  in  which 
the  bites  are  on  the  head,  are  always  more  serious,  and  the  mortal- 
ity is  higher.  Like  tetanus  the  virus  travels,  it  is  supposed,  from 
the  site  of  injury  to  the  central  nervous  system  by  way  of  the 
nerves.  If  the  bite  was  on  the  toe,  it  would  take  longer  for 
infection  to  reach  the  brain,  than  if  it  was  on  the  upper  lip.  This 
is  a  very  plausible  explanation  of  the  varying  incubation  periods 
in  both  tetanus  and  hydrophobia. 

Coley's  Fluid  in  the  Treatment  of  Tumors 

This  method  of  treatment  is  in  no  wise  a  prophylactic  one,  but 
strictly  a  curative  one.  It  consists  in  the  injection  of  the  toxins 
of  streptococci,  in  the  hope  that  they  will  cause  a  shrinking,  or  dis- 


OPSONINS    AND   OPSONIC   INDEX  89 

appearance  of  malignant  sarcomata.  An  attack  of  erysipelas  (it 
has  long  been  observed)  occurring  in  a  patient  with  some  malig- 
nant disease,  has  the  effect  of  causing  a  disappearance,  or  retro- 
gression, of  the  tumors.  Artificial  infection  with  streptococci 
was  then  practiced  with  the  idea  that  it  might  produce  the  same 
effect.  But  this  was  found  to  be  dangerous.  Coley  prepared 
toxins  of  streptococci  by  allowing  them  to  grow  with  the  B.  Pro- 
idgiosus.  The  mixture  after  a  Jong  period  of  incubation  was  steril- 
ized by  heat,  and  the  fluid  thus  obtained  was  injected  into  the 
tissues.  Virulent  strains  of  streptococci  are  used  and  the  dose 
of  the  dead  culture  is  about  half  a  drop  given  under  strict  anti- 
septic precautions.  The  best  results  are  obtained  in  spindle- 
cell  sarcoma,  and  the  poorest  in  the  melanotic  variety.  The 
method  by  no  means  should  be  employed  where  the  tumor  can 
be  removed  by  operation.  It  cannot  supplant  the  knife,  and 
only  in  inoperable  cases  or  as  a  supplementary  treatment  where 
other  forms  of  treatment  are  employed,  should  it  be  used. 

Opsonins  and  Opsonic  Index 

Peculiar  substances  in  blood  serum  have  been  called  by  Wright  and  Doug- 
lass opsonins  (Greek:  prepare  food  for) .  If  fresh  blood  is  mixed  with  an  emul- 
sion of  some  bacteria  and  then  incubated  for  half  an  hour,  it  will  then  be 
found  that  many  of  the  bacteria  are  within  the  polymorphonuclear  leucocytes. 
If  the  serum  is  washed  away  from  the  leucocytes  before  adding  bacteria,  none 
of  the  latter  will  be  found  within  the  leucocytes.  This  proves  that  the  serum 
has  some  influence  on  phagocytosis.  In  order  to  show  that  this  effect  is  on 
the  bacteria  rather  than  on  the  leucocytes,  the  bacterial  suspension  may  be 
treated  with  some  serum  for  half  an  hour  and  then  washed  free  from  this 
serum  by  means  of  a  salt  solution  in  a  centrifuge,  and  then  mixed  with  some 
serum-free  leucocytes;  then  it  will  be  found  that  phagocytosis  occurs  as  before. 
The  bacteria  have  been  "sensitized."  According  to  Wright  this  action  is 
comparable  to  cooking  or  peptonizing. 

Phagocytosis  then  depends  upon  the  action  of  serum  upon  bacteria, 
which  are  coped  with  in  the  body,  first  by  the  action  of  the  serum,  and  then 
by  the  leucocytes.  It  is  thermostabile. 

The  quantitative  action  of  phagocytosis  may  be  estimated  by  Leishman's 
method.  He  mixed  blood  and  an  emulsion  of  bacteria  in  salt  solution  in  equal 


QO  IMMUNITY 

quantities,  and  allowed  them  to  stand  for  thirty  minutes  in  the  incubator. 
After  this  the  mixture  was  stained  and  the  average  number  of  bacteria  per 
leucocyte  was  obtained.  The  result  was  known  as  the  phagocytic  index. 

Wright  has  devised  the  following  technique.  Young  cultures,  a  few  hours 
old,  are  employed.  These  are  scraped  off  agar  tubes  and  mixed  with  salt 
solution.  After  this  has  sedimented,  the  supernatant  fluid  is  separated  from 
the  bacterial  masses  by  a  centrifuge;  is  pipetted  off,  and  preserved. 

Washed  leucocytes  are  obtained  by  collecting  2  c.c.  of  blood  in  30  c.c.  of 
salt  solution  containing  i  percent  citrate  of  soda  to  prevent  blood  coagula- 
tion. The  serum  and  citrate  of  soda  are  separated  from  corpuscles  by 
washing  twice  in  a  centrifuge.  The  upper  layer  of  the  sediment  is  rich  in 
washed  leucocytes,  and  is  used  in  the  experiments. 

To  obtain  the  opsonic  index,  blood  serum  from  various  cases  is  collected. 
In  the  case  of  staphylococcus  infection — say  furuncle — the  blood  serum  is 
drawn  from  the  patient  and,  with  equal  portions  of  an  emulsion  of  staphylo- 
cocci  (young  culture),  and  a  suspension  of  washed  corpuscles,  is  thoroughly 
mixed  in  a  pipette,  which  after  the  ends  are  sealed,  is  placed  in  an  incubator 
for  fifteen  minutes.  A  drop  of  the  mixture  is  then  spread  upon  a  slide;  fixed, 
and  stained  with  Jenner's  stain.  The  number  of  staphylococci  in  50  poly- 
nuclear  leucocytes  is  determined  and  divided  by  50  to  obtain  the  average. 

At  the  same  time  that  this  experiment  is  being  performed,  some  normal 
serum  should  be  used  in  another  experiment;  an  emulsion  of  staphylococci 
and  washed  leucocytes  being  used  as  above.  After  pursuing  the  same  steps 
in  this  experiment  as  in  the  first,  the  average  number  of  staphylococci  per 
leucocyte  is  determined. 

Tq  obtain  the  opsonic  index,  it  is  necessary  to  know  the  ratio  of  staphylo- 
cocci in  the  leucocytes  treated  with  furuncular  serum  and  with  normal  serum. 
If  the  normal  serum  leucocytes  contained  10  staphylococci,  and  the  furun- 
cular serum  contained  15,  the  index  would  be  1.5. 

In  the  case  of  tubercle  bacilli,  the  latter  must  be  heated  to  ioo°C.  to  kill 
them,  otherwise  they  will  be  agglutinated  by  the  serum,  and  a  homogeneous 
emulsion  will  not  be  obtained.  After  heating,  the  clumps  must  be  broken  up 
by  grinding  the  masses  in  an  agate  mortar,  adding  a  little  salt  solution  from 
time  to  time  until  the  mass  is  thoroughly  broken  up.  The  bacilli  must  then, 
after  phagocytosis,  be  stained  by  carbol  fuchsin  and  decolorized  with  acid 
alcohol.  If  the  leucocytes  are  left  too  long  in  contact  with  the  organisms 
they  may  become  so  engorged  as  to  prevent  counting,  the  number  increasing 
from  5.7  percent  after  five  minutes  to  28.5  percent  in  two  hours. 

Highly  immunized  anti-bacterial  serums  have  much  greater  opsonic  powers 
than  have  normal  ones,  anti-streptococcus  and  anti-pneumococcus  sera 
being  especially  pwerful  toward  streptococci  and  pneumococci.  It  is  pos- 


OPSONINS    AND   OPSONIC   INDEX  91 

sible  to  increase  the  opsonic  powers  of  the  blood  of  an  individual  suffering 
from  an  infection,  by  vaccinating  him  with  killed  cultures  of  the  organism 
with  which  he  was  infected. 

The  determination  of  the  opsonic  index  is  a  long  and  tedious  affair  so  that 
in  practice  it  is  only  used  when  it  is  necessary  to  estimate  the  value  of  the 
vaccine  treatment  or  to  control  dosage.  Under  ordinary  circumstances 
clinical  phenomena  will  indicate  the  correctness  of  dosage  and  interval 
but  certain  cases  that  fail  to  do  well  should  be  checked  by  opsonin  indicators. 
The  value  rises  slowly  and  steadily  with  appropriate  vaccine,  dosage  and 
interval,  falls  with  too  large  quantity,  shows  no  change  with  inadequate 
quantities. 

The  Local  Reactions  or  Tests. — We  have  learned  in  the  past  few  years 
that  the  skin  and  mucous  membranes  will  react  more  or  less  specifically  to 
the  bacterial  proteins.  It  is  a  form  of  allergic  (see  page  66).  There  have 
been  developed  local  tests  for  tuberculosis,  syphilis,  typhoid,  glanders  and 
other  diseases.  The  first  two  being  the  most  important  are  considered  below. 
The  others  are  of  similar  nature. 

Tuberculosis. — If  tuberculin  of  any  form  be  rubbed  into  an  abraded  skin 
area  (Von  Pirquet's  cutaneous)  or  injected  between  the  layers  (Moro's'per- 
cutaneous)  of  the  skin  a  red  maculopapule  or  even  vesicle  upon  an  inflamed 
base  will  appear  within  twenty-four  hours.  There  may  be  a  mild  general 
reaction  of  fever  and  malaise.  A  positive  reaction  to  such  an  installation 
simply  indicates  the  presence  of  a  tuberculous  lesion  and  that  an  allergic 
fstate  of  the  skin  exists  but  does  not  show  whether  or  not  the  lesion  is  active. 
For  this  reason  it  is  only  of  value  in  children  since  three-fourths  of  adults 
are  believed  to  have  a  healed  lesion  within  them.  Not  only  upon  the  skin 
but  upon  the  conjunctiva  can  this  reaction  be  obtained. 

Syphilis. — The  poison  of  the  Treponema  pallidum  is  called  luetin.  It  is 
made  by  grinding  up  in  salt  solution  a  culture  of  the  germ,  heating  the  result- 
ing mass  to  6o°C.  for  an  hour  and  preserving  it  with  phenol.  If  this  be  in- 
stilled into  an  abraded  skin  area  a  maculopapule  or  nodular  eruption  occurs 
in  a  syphilitic.  This  positive  outcome,  however,  appears  only  in  late  cases, 
those  of  tertiary  stages  and  in  treated  cases.  It  therefore  complements  the 
Wassermann  reaction,  being  positive  where  this  is  apt  to  fail. 

Schick  Test. — It  has  been  found  by  Schick  and  others  that  if  Ko  minimum 
lethal  dose  of  diphtheria  toxin  in  .2  c.c.  of  saline  be  injected  into  the  skin  of 
a  person,  a  swollen,  pink,  tender  area  will  appear  in  persons  susceptible  to 
infection  with  Klebs-LofHer  bacilli.  If  no  such  reaction  occurs,  the  person 
is  not  susceptible.  This  depends  upon  the  fact  that  if  anti-toxin  be  present 
in  the  blood  it  will  combine  with  the  toxin  and  no  reaction  will  appear,  while 
if  no  anti-toxin  be  present  the  toxin  is  free  to  exert  its  effect.  It  has  been 


Q2  IMMUNITY 

determined  that  persons  giving  a  negative  reaction  need  not  receive  immuniz- 
ing doses  of  anti-toxin.  About  80  percent  of  adults  and  30  to  40  percent 
of  children  are  immune.  A  negative  reaction  seems  to  indicate  that  the  blood 
contains  Ko  c.c.  more  unit  of  anti-toxin  per  cubic  centimeter. 

Isoagglutinin  and  Isohemolysin 

There  are  frequently  in  the  blood  of  animals,  ly tic  and  agglutina- 
tive anti-bodies  for  the  red  cells  of  other  members  of  the  same 
species.  By  this  is  meant  that  if  the  blood  of  a  man  be  mixed  with 
that  of  another  the  cells  of  one  of  them  may  be  clumped  or  dis- 
solved. This  is  of  considerable  importance  to  the  surgeon  who 
wishes  to  transfuse  blood,  for  were  he  to  use  a  donor  whose  blood 
was  unlike  that  of  the  recipient  the  latter  would  have  a  serious 
chill,  indications  of  blood  destruction  and  shock,  and  he  might  die. 

These  two  substances  are  probably  independent  in  action  but 
are  so  commonly  found  acting  in  harmony  that  they  may  be  con- 
sidered inter-dependent.  Landsteiner  and  Jansky  divided  persons 
into  four  groups  according  to  their  agglutinin  and  agglutinogen. 
Moss  made  the  same  observation  but  classified  them  differently. 

JANSKY'S  GROUPING 

Serum 
Cells  I  II  III  IV  Percent 

I  42.84 

II  +  +  10.36 
HI                         +                  +                                                              41-38 
IV                          +                  +                  +  5-42 

This  means  that  the  cells  of  Type  I  are  not  agglutinated  by  an] 
of  the  serums,  whereas  the  cells  of  Type  IV  are  clumped  by 
serums  but  their  own. 

Agglutination  is  accomplished  according  to  Landsteiner  by  tw< 
agglutinins  a  and  b  which  are  both  contained  in  serums  clumping 
all  type  of  corpuscles,  whereas  both  are  absent  in  serums  clumpii 
no  cells.  Agglutinable  bodies,  represented  by  a  and  b,  are  suppos 
to  be  present  in  the  respective  cells  which  can  be  clumped,  absent 
in  the  others.  Group  I  therefore  contains  agglutinins  A  and  B 
but  does  not  contain  agglutinable  bodies  a  and  b.  Other  groups 


ISOAGGLUTININ   AND   ISOHEMOLYSIN  93 

would  be  explained  on  the  same  basis.  While  Jansky's  methods 
have  priority  in  time  of  publication  the  American  surgeon  has 
during  the  great  war  become  accustomed  to  the  Moss  grouping 
which  is  as  follows : 

Serum 
Cells  I  II  III  IV 

I  +  +  + 

II  +  + 
HI                                                            +                                                        + 
IV 

It  will  be  seen  that  this  is  merely  the  reverse  of  the  first  one  and 
that  Type  IV  has  agglutinins  but  no  agglutinable  bodies. 

Agglutinins  and  agglutinable  bodies  may  be  absorbed  from  a 
given  blood  by  saturation  with  their  respective  antigen. 

In  transfusion,  homologous  bloods  should  be  mixed,  but  groups 
I  or  IV  respectively  may  be  considered  as  universal  donors  since 
experience  shows  it  safe  to  use  a  blood  which  agglutinates  the 
recipient's  cells.  It  is  however,  incorrect  to  use  a  blood  whose 
corpuscles  are  clumped  by  the  recipient's  serum.  The  agglutina- 
tive titer  of  human  blood  is  not  very  high,  i-io  to  1-25,  and  as 
the  entering  serum  is  diluted  the  agglutination  titer  would  be 
exceeded. 

Further  experiments  on  the  independence  of  the  two  anti-bodies 
would  indicate  that  usually  agglutination  precedes  hemolysis  but 
this  need  not  be  so,  in  a  small  percentage  of  cases.  For  practical 
purposes  agglutination  tests  are  sufficient  indications  of  the  com- 
patibility of  bloods.  Tests  for  compatibility  take  the  form  'of 
(i)  direct  mixture  of  the  blood  of  recipient  and  of  donor  and  (2) 
the  testing  of  donor's  blood  by  standard  serums. 

i.  Patient's  blood  A — serum  separated  from  the  clot.     B— 
cells  from  defibrinated  blood  washed  in  saline  and  resuspended  in 
saline.     Donor's  blood  a — serum  separated  from  the  clot,  b — cells 
from  defibrinated  blood  washed  in  saline  and  resuspended  in  saline. 
Test  A — 4  parts  b  i  pt. 
a — 4  parts  B  i  pt. 
Incubate  at  37°  for  one  hour. 


94  IMMUNITY 

If  bloods  be  compa table  no  change  will  occur  in  the  blood  cells. 
If  donor's  corpuscles  be  agglutinated  by  recipient's  serum  there 
will  be  clumping  in  the  first  tube;  if  the  reverse,  a  change  will 
occur  in  the  second  tube.  This  method  while  often  used  is  more 
trouble  and  no  more  reliable  than  the  following : 

2.  It  is  necessary  to  have  for  the  group  determinations,  known 
sera  of  the  various  types,  but  for  practical  purposes  types  II  and 
III  will  reveal  any  type  corpuscles  suspended  in  them.  The  blood 
to  be  tested  is  caught  from  a  prick  in  the  finger  into  a  few  drops  of 
sodium  citrate  solution.  One  drop  of  this  cell  suspension  is  mixed 
with  one  drop  of  type  II  and  of  type  III  sera  separately  on  a  slide. 
After  a  period  of  five  to  thirty  minutes  clumping  may  occur. 
If  it  occur  in  both,  the  blood  is  by  the  Moss  scale  group  I;  if  in 
neither  it  is  group  IV;  if  clumping  occur  in  serum  III  and  not  in 
serum  II,  the  blood  is  group  II;  if  clumping  occur  in  group  II 
and  not  in  group  III  the  blood  is  group  III. 


CHAPTER  V 
STUDY  OF  BACTERIA 

Bacteria  are  studied  in  the  following  various  ways: 

1.  Morphological  characteristics,  form,  size,  motility,  presence 
of  spores,  granules,  capsules,  and  flagella.     Reaction  of  proto- 
plasm to  dyes  and  reagents. 

2.  Characteristics  of  growth  in  culture  media;  appearances  of 
culture;  chemical  activities;  production  of  acid,  gases,  toxins, 
colors,  etc.;  reactions  to  heat,  disinfectants,  light,  etc. 

3.  Study  of  the  action  of  bacteria  on  the  tissues  of  man  and 
animals,  and  of  the  toxins  on  the  tissues  and  functions  of  the 
various  organisms. 

The  simplest  way  to  study  bacteria  is  to  make  a  hanging  drop  of 
a  fluid  containing  bacteria,  and  observing  the  organisms  under  a 
microscope.  To  do  this,  a  cover-slip,  and  a  slide  with  a  concavity 
ground  in  it  are  used.  A  drop  of  bacteria  laden  fluid  is  placed  on 
the  cover-glass,  and  after  the  edges  have  been  smeared  with  vase- 
line, the  cover-slip  is  inverted  over  the  concavity  in  the  slide,  and 
the  bacteria  can  then  be  examined  with  either  the  dry  %  inch,  or 
the  one-twelfth  oil  immersion  objective.  If  the  preparation  is 
kept  warm  for  some  time,  various  vital  phenomena  may  be  noted. 
Direct  division,  sporulation,  motility,  agglutination,  and  bacterio- 
lysis can  be  studied  by  this  means.  Instead  of  using  a  fluid,  a 
block  of  nutrient  agar  may  be  cemented  to  the  cover-glass;  after 
the  bacteria  have  been  planted  on  the  agar,  the  various  vital 
phenomena  may  be  noted. 

All  minute  bodies,  whether  they  be  bacteria,  dust  particles  or 
granules  of  india  ink  in  suspension,  exhibit  a  trembling  vibrating 
motion  called  the  Brownian  motion.  Motile  bacteria  either  move 

95 


96  STUDY   OF  BACTERIA 

so  swiftly  that  the  eye  can  hardly  follow  them,  or  they  may  merely 
roll  or  wiggle  across  the  field  slowly.  Direct  division,  if  pro- 
ceeding under  the  best  conditions,  requires  but  fifteen  to  forty 
minutes.  It  is  best  observed  in  a  warm  stage  or  when  working  in 
a  room  kept  at  a  temperature  of  35°C.  Sporulation  occurs  differ- 
ently in  different  species.  In  some  it  will  be  found  soon  after  the 
culture  has  been  removed  from  the  incubator,  while  in  others 
several  hours  are  required.  Sporulation,  it  must  be  remembered 
is  a  resistant  stage  when  unfavorable  conditions  are  met. 

The  Gruber-Widal  reaction  is  thus  studied.  A  drop  of  the 
serum  and  bullion  culture,  mixed  in  proper  proportions,  is  dropped 
on  a  cover-slip,  which  is  then  placed,  drop  downward,  over  the 
cavity  of  the  slide  (hanging  drop,  Fig.  20)  (see  Agglutination) . 


FIG.  20. — Hanging  drop,  over  hollow  ground  slide.     (Williams.) 

Staining  bacteria  is  a  matter  that  is  easily  accomplished,  and 
very  many  staining  solutions  and  methods  have  been  invented  for 
this  purpose. 

The  simplest  procedure  is  to  take  a  drop  of  pus,  blood  or  culture, 
and  spread  it  upon  a  very  clean  slide  with  a  sterilized  platinum 
needle.  The  matter  must  be  spread  thinly  and  evenly.  After  the 
water  has  evaporated  and  the  preparation  has  become  dry  without 
the  use  of  Jieat,  it  must  be  fixed.  To  do  this  various  agents  are 
used.  The  object  of  the  fixing  is  to  coagulate  the  protoplasm  of 
the  cells,  and  to  fasten  all  the  smeared  matter  fast  to  the  glass, 
so  that  the  staining  fluid  and  water  will  not  wash  them  off.  This 
is  accomplished,  for  bacteria  usually,  by  holding  the  smeared  slide 
in  the^apex  of  a  bunsen  flame  until  quite  warm  to  the  hand.  Great 
care  must  be  used  not  to  char  the  film.  Experience  is  need( 
to  fix  slide  smears  correctly.  The  beginner  would  do  well  t( 
use  cover-slips.  If  a  cover-slip  is  used  it  must  be  passed  througl 
the  flame  three  times  rapidly.  After  fixing  and  thorough  cooling, 


STAINING  BACTERIA  97 

the  staining  fluid  is  poured  on,  and  after  remaining  a  few  minutes 
is  poured  off  and  the  slide  is  washed,  dried  by  blotting  paper, 
and  examined.  If  a  cover-slip  has  been  used  a  drop  of  balsam  is 
put  upon  a  clean  slide  and  the  cover,  smeared  with  stained 
bacteria,  is  inverted  on  the  balsam.  Upon  the  stained  bacteria 
themselves  (if  a  cover-glass  has  not  been  used)  or  upon  the  cover- 
slip  a  drop  of  cedar  oil  may  be  placed,  and  the  preparation 
examined  with  a  one-twelfth  objective.  This  is  one  of  the 
simplest  staining  procedures  practised  in  bacteriology.  Other 
more  complicated  methods  will  now  be  described. 

Besides  heat,  absolute  alcohol,  methyl  alcohol,  or  formalin  may 
be  used  as  fixatives.  Some  stains  are  made  up  with  methyl 
alcohol,  and  instead  of  fixing  by  heat,  the  stain  is  merely  dropped 
upon  the  dried  film,  and  the  bacteria  are  fixed  and  stained  by  the 
same  solution  at  the  same  time,  water  being  added  for  differentia- 
tion at  the  end. 

Aniline  dyes  are  almost  entirely  used  as  stains  in  bacteriology 
and  these  are  divided  into  two  classes,  the  basic  and  acid  stains, 
according  as  their  staining  properties  depend  upon  the  basic  or 
acid  part  of  the  molecule.  Basic  dyes  stain  nuclear  tissues  of  cells 
and  bacteria.  The  acid  are  used  as  contrast  stains  and  do  not 
color  bacteria,  but  tissues  in  which  they  may  be  imbedded. 

The  common  basic  stains  are  methyl  violet,  and  gentian  violet, 
methyl  green,  methyl  blue,  and  methylene  blue,  thionin  blue,  Bis- 
marck brown,  fuchsin,  and  saffranin.  These  are  used  for  staining 
different  bacteria  under  different  conditions.  The  most  useful 
stain  is  methylene  blue,  since  it  is  difficult  to  overstain  with  it,  and 
it  is  very  easily  applied.  It  has  been  found  that  certain  physical 
and  chemical  conditions  are  necessary  for  successful  staining  with 
aniline  dyes.  Alcoholic  solution  of  dyes  entirely  devoid  of  water 
do  not  stain;  absolute  alcohol  does  not  decolorize  bacteria  after 
staining  with  aniline  colors,  while  diluted  alcohol  decolorizes 
readily.  The  more  completely  a  dye  is  dissolved,  the  weaker  is  its 
staining  power.  A  dyestuff  unites,  as  a  whole,  with  the  bacterial 

7 


98  STUDY   OF  BACTERIA 

plasm,  forming,  as  it  were,  a  double  salt  between  the  two.  Cer- 
tain substances,  alkalies,  carbolic  acid,  iron  and  copper  sulphate, 
tannic  acid,  alum,  and  aniline  oil,  are  added  to  a  solution  of  aniline 
dyes,  and  they  act  as  mordants,  or  fixatives,  making  the  dye  bite 
into  the  protoplasm  of  the  bacterial  cells.  Spores,  capsules,  and 
flagella,  are  hard  to  stain,  and  special  heavily  mordanted  stains  are 
used  to  demonstrate  them.  Chemical  reaction  occurring  in  the 
cell  protoplasm  is  of  great  value  in  differentiating  bacteria.  The 
presence  of  granules  in  bacterial  cells  is  often  only  shown  by 
the  use  of  special  stains,  which  deeply  color  them.  Bacteria  of  the 
tubercle  group  are  called  "acid-fast,"  because,  after  being  stained, 
it  is  difficult  to  decolorize  them  with  acid  solutions.  These  bac- 
teria are  hard  to  stain  and  resist  decolorizing  agents  after  they 
are  stained. 

1 .  Loffler's  alkaline  methylene  blue  solution  consists  of 

Saturated  alcoholic  solution  of  methylene  blue 30  c.c. 

Ho, ooo  solution  caustic  soda  solution  in  water 100  c.c. 

Mix. 

This  is  the  most  useful  of  all  the  staining  mixtures  employed. 

2.  ZeihFs  solution  carbol-fuchsin  consists  of 

Fuchsin i  gram. 

Carbolic  acid  crystals 5  grams. 

Dissolved  in  100  c.c.  of  water,  to  which  is  added  10  c.c.  of  absolute  alcohol. 

This  can  also  be  made  by  taking  a  5  percent  solution  of  carbolic 
acid  in  water  and  adding  sufficient  saturated  solution  of  fuchsin  in 
water  until  a  bronze  scum  persists  upon  the  top.  This  is  used  for 
staining  tubercle  bacilli  in  sputum  and  sections.  It  must  be 
heated  when  used  for  rapid  staining.  Tubercle  bacilli  can  be 
stained  in  cold  solution,  if  immersed  over  night  in  it. 

3.  Fuchsin  solution. 

Saturated  alcoholic  solution  of  basic  fuchsin i  c.c. 

Water. . .  .   100  c.c. 


STAINING  BACTERIA  99 

4.  Bismarck  brown  solution. 

Water 100  c.c. 

Bismarck  brown  sufficient  to  saturate. 
Filter  and  use  a  contrast  stain. 

5.  Weigert's  aniline  gentian  violet  stain. 

Gentian  violet i  gram. 

Dissolve  in  absolute  alcohol 15  c.c. 

Distilled  water 80  c.c. 

Then  add  to  this 

Aniline  oil 3  c.c. 

Mix,  shake  and  filter. 

This  stain  can  also  be  prepared  by  taking  a 

Sat.  watery  solution  of  aniline  oil 100  c.c. 

Filter,  then  add 

Sat.  alcoholic  solution  gentian  violet 10  c.c. 

Sterling's  permanent  solution  is  made  by  mixing  2  c.c.  of  aniline  oil  with 
10  c.c.  of  95  percent  alcohol;  the  mixture  is  shaken  and  88  c.c.  of  distilled 
water  added;  5  grams  of  gentian  violet  powdered  in  a  mortar  receives  the 
above  fluid,  added  slowly  while  grinding;  filter.  This  solution  while  expen- 
sive to  make,  requires  only  a  sm,all  quantity,  stains  rapidly  and  keeps  well. 

This  is  a  very  intense  bacterial  stain  used  for  demonstrating 
bacteria  by  the  Gram  method. 

Gram's  method  of  staining. 

A  cover-glass  is  spread  with  a  smear  of  bacteria,  or  pus  to  be 
examined.  After  air-drying  it,  and  fixing  it  in  the  flame,  the  ani- 
line gentian  violet  is  poured  on,  allowed  to  stand  for  three  minutes, 
then  poured  off  and  the  preparation  treated  with 

Iodine  crystals i  gram. 

Potassium  iodide 2  grams. 

Water 100  c.c. 

for  two  minutes.  This  renders  the  purplish  preparation  grayish 
in  appearance.  Alcohol  is  now  poured  upon  the  preparation  re- 
peatedly until  the  alcohol  does  not  dissolve  any  more  color.  A 
contrast  stain  of  Bismarck  brown  or  dilute  fuchsin  or  safranin  is 


100  STUDY   OF  BACTERIA 

now  used.  If  the  bacteria  on  examination  remain  a  dark  violet- 
blue  they  are  then  said  to  stain  by  Gram's  method,  or  are  "  Gram- 
positive."  If  they  are  decolorized  they  take  the  contrast  stain  and 
are  said  not  to  stain  by  this  method,  and  are  "Gram-negative. " 

Many  bacteria  stain  in  this  way,  and  many  do  not.  Important 
bacteria  often  may  be  differentiated  in  this  manner. 

Examples  of  Gram's  stain  are  as  follows: 

Gram-positive — Bact.  aero  genus  capsulatus,  Bact.  anthracis, 
Bact.  diphtheria,  B.  tetani,  Bact.  tuberculosis,  Streptococcus 
pneumonia,  Staph.  pyogenes,  Strep,  pyogenes.  Gram-negative — 
B.  coli,  B.  dysenteries,  Bact.  influenza,  Bact.  mallei,  Bact.  pestis, 
B.  pyocyaneus,  B.  typhosus,  Diplococcus  intracellularis  menin- 
gitidis,  Micr.  catarrhalis,  Micr.  gonorrhoea,  Spirillum  cholera. 

Thionin  Blue,  or  Carbol  Thionin 

This  is  a  useful  stain,  prepared  thus: 

Thionin  blue i      gram. 

Carbolic  acid !       2.5  grams. 

Water 100      c.c. 

Filter.     Good  for  staining  bacteria  in  tissues. 

Special  Stains 

Wright's  Stain. — This  not  only  stains,  but  fixes.  It  has  a  wide 
range  of  usefulness  in  a  bacteriological  laboratory  for  the  staining 
of  blood,  pus,  malarial  parasites,  trypanosomes,  as  well  as  many 
bacteria,  and  is  prepared  as  follows : 

.5  percent  solution  of  sodium  bicarbonate 100  c.c. 

Methylene  blue i  gram. 

Mix  and  heat  in  sterilizer  one  hour  at  ioo°C.  Cool,  filter,  then  mix  Ko  per- 
cent yellowish  eosin  in  water  until  the  mixture  loses  its  blue  color  and  becomes 
purplish.  Of  the  eosin  solution  add  500  c.c.  to  each  100  c.c.  of  the  methylene 
blue  mixture.  Mix  and  collect  the  abundant  precipitate  which  immediately 
forms  on  a  filter.  Dry  this  and  dissolve  in  methyl  alcohol  in  the  proportion 
of  i  gram  of  powder  to  600  c.c.  of  the  alcohol.  This  is  the  staining  fluid. 
Keep  well  stoppered.  Fresh  alcohol  may  be  added  for  that  which  evaporates. 


SPECIAL   STAINS  '  IOI 

This  complex  stain  represents -a  type  *ofv  which  Jenner's,  Leish- 
man's,  and  Romanowsky's  are  members.  To  use  this  stain,  a 
blood  or  pus  film  is  spread  and  air-dried.  The  stain  is  then  run 
on  the  slip,  or  slide,  for  one  minute.  After  this  time  slowly  drop 
distilled  water  in  quantity  similar  to  that  of  stain  used.  This  is 
when  the  true  staining  takes  place.  After  three  minutes  wash  in 
distilled  water,  dry  and  mount.  Nuclei,  malarial  parasites,  try- 
panosomes,  and  bacteria  are  stained  blue;  red  cells  are  stained 
pinkish-orange,  while  the  granules  of  the  leucocytes  are  stained 
pink,  lilac,  or  blue,  depending  upon  their  character. 

Giemsa's  Stain 

This  stain  is  used  for  demonstrating  the  organism  of  syphilis, 
trypanosomata,  granules  and  the  like,  and  is  prepared  as  follows : 

Azur  II  Eosin 3  grams. 

Azur  II 8  grams. 

Glycerine  C.  P 250  c.c. 

Methyl  alcohol 250  c.c. 

Bacteria  are  often  covered  with  capsules  that  are  difficult  to 
stain,  and  special  methods  have  been  devised  to  demonstrate 
them. 

1.  Air-dry  the  specimen. 

2.  Harden  and  fix  in  absolute  methyl  alcohol. 

3.  Dilute  stain  with  distilled  water,  using  one  drop  of  stain  to  each  cubic 
centimeter  of  water. 

4.  Cover  preparation  with  dilute  stain  fifteen  minutes  to  three  hours 
(longer  period  for  spirochaetesj. 

5.  Wash  in  running  water. 

6.  Blot  and  mount. 

Capsule  Staining 
Welch's  Method. 

1.  Cover-glass  preparations  are  made  in  the  usual  manner,  and  over  the 
film  after  fixing,  glacial  acetic  acid  is  poured. 

2.  Without  washing  off  the  acid,  aniline  water  gentian  violet  is  poured  on. 


102  ST?JE\    OF  BACTERIA 

Charge  the  stain  four  or  five  times  to  remove  the  acid.  Stain  four 
minutes,  and  wash  with  2  percent  NaCl  solution,  not  water.  This 
demonstrates  the  capsule  very  well. 

His's  Method. 

"A." 

1.  Make  cover-glass  preparation  mixing  specimen  with  blood  serum.     Fix 
in  flame. 

2.  Stain  for  a  few  seconds  with  a  half  concentrated  water  solution  of  gentian 
violet. 

3.  Wash  in  weak  potassium  carbonate  solution  for  a  few  minutes. 

4.  Dry  and  mount. 
"B." 

.   2.  Dry  and  fix. 

3.  Heat  and  pour  on  the  following  stain,  steaming  thirty  seconds: 

(a)  Saturated  alcoholic  solution  of  gentian  violet 5  c.c. 

(b)  Water 95  c.c. 

4.  Wash  in  a  20  percent  solution  cupric  sulphate. 

5.  Dry  and  mount. 

Spore  Staining 

Spores  resist  stains,  and  when  stained  are  hard  to  decolorize. 

1.  Dry  and  fix  in  the  usual  way. 

2.  Flood  cover-glass  with  hot  carbol-fuchsin;  heat  until  it  steams;  repeat 
this  once  or  twice.    This  stains  bacteria  and  spores. 

3.  Wash  in  water. 

4.  Decolorize  with 

Alcohol 2  parts. 

i  percent  acetic  acid i  part. 

5.  Wash. 

6.  Counterstain  with  methylene  blue. 

7.  Wash,  dry  and  mount. 

By  this  method,  which  is  a  simple  and  satisfactory  one,  the 
spores  are  stained  a  brifliantjred,  while_the  body  of  the  bacilli 
are  stained  blue. 

Flagella  Staining 

To  a  beginner  flagella  staining  is  difficult;  there  have  been  many 
well-known  methods  devised.  The  simpler  are  as  effective  as  the 
more  complicated  but  do  not  always  make  as  pretty  preparations. 


FLAGELLA   STAINING  103 

Flagella,  being  processes  extending  from  the  capsule,  are,  like 
the  latter,  hard  to  demonstrate.  They  are  not  stained  by  the 
common  bacterial  stains.  In  general  a  powerful  stain  mixed  with 
a  strong  mordant  must  be  employed.  Some  methods  appear 
to  be  not  so  much  a  staining  method  in  the  ordinary  sense  but 
either  a  precipitaing  of  the  stain  in  the  substance  of  the  flagella 
or  else  a  decomposition  of  silver  salts  in  the  flagella  substance. 
To  stain  flagella,  a  young  culture  grown  on  agar  must  be  employed ; 
glycerine  agar  must  never  be  used.  A  mass  of  the  organism  is 
gently  mixed  with  a  drop  of  distilled  water  until  a  uniform  emul- 
sion is  made.  A  dozen  cover-slips  carefully  washed  and  cleaned 
by  alcohol  are  thoroughly  flamed  in  order  to  remove  the  slightest 
trace  of  grease.  The  watery  emulsion  of  bacteria  is  then  spread 
over  the  cover-slips  evenly  and  thinly.  After  they  are  dry  the 
bacteria  are  fixed  by  holding  them  for  a  minute  just  above  the 
apex  of  the  flame  with  the  fingers.  The  following  methods  may  be 
pursued : 

Pitfield's  Method  Modified  by  Muir. 

Two  solutions  are  necessary  for  this  method. 

A.  Mordant. 

10  percent  watery  solution  tannic  acid '. . .   10  c.c. 

Corrosive  sublimate  saturated  water  solution 5  c.c. 

Carbol-fuchsin  solution 5  c.c. 

This  forms  a  dense  precipitate  which  must  be  removed  by  the  centrifuge, 
or  sedimentation,  and  the  clear  fluid,  or  mordant,  is  stored  in  a  bottle.  It 
keeps  for  two  weeks. 

B.  Stain. 

Saturated  watery  solution  of  alum 10  c.c. 

Saturated  alcoholic  solution  gentian  violet 2  c.c. 

This  keeps  but  two  or  three  days. 

Flood  the  cover-slip  with  the  mordant  and  gently  steam  for  one 
minute,  then  wash  and  dry  thoroughly,  pour  the  stain  on  and 
steam  for  one  minute  more.  Wash,  dry  and  mount. 

This  method  yields  very  good  results. 


104  STUDY   OF  BACTERIA 

Pitfield's  Method. 

This  is  the  simplest  stain  and  the  easiest  to  use,  but  does  not 
give  the  good  results  that  the  previous  one  does.  But  one  solution 
is  needed,  this  is  made  in  two  parts  and  mixed. 

A.  Tannic  acid i  gram. 

Water 10  c.c. 

B.  Saturated  watery  solution  alum  (old) 10  c.c. 

Saturated  alcoholic  solution  gentian  violet i  c.c. 

Mix. 

A  heavy  precipitate  is  formed  by  this  process  which  is  useful  in  the  stain- 
ing. The  stain  is  almost  a  saturated  solution  of  alum  and  tannic  acid,  and 
when  it  becomes  supersaturated  by  evaporation  and  heat,  staining  takes  place. 
After  this  the  process  is  very  simple.  The  cover-slip  is  carefully  flooded  with 
the  stain  and  warmed  for  a  minute  over  the  flame  of  a  bunsen  burner,  turned 
very  low,  until  steam  arises.  Not  too  much  stain  should  be  run  over  the 
cover-slip.  After  steaming  occurs,  the  stain  should  remain  for  a  minute,  then 
the  preparation  is  washed,  dried  and  mounted.  It  will  be  found  that  the  best 
stained  flagella  are  on  those  bacteria  nearest  to  the  edges  where  the  evapora- 
tion has  been  most  intense.  If  the  preparation  is  not  equally  stained,  Wei- 
gert's  aniline  gentian  violet  can  be  run  on  for  a  minute  to  deepen  the  color. 

Loffler's  Method. 

This  is  the  original  flagella  stain  and  is  a  very  good  one. 
It  is  made  as  follows : 

A.  Mordant 

20  percent  watery  solution  tannic  acid 10  c.c. 

Sat.  solution  ferrous  sulphate 5  c.c. 

Fuchsin  sat.  alcoholic  solution i  c.c. 

Mix. 

B.  Stain 
Carbol-fuchsin. 

Proceed  as  in  the  previous  methods. 

The  most  important  steps  in  flagella  staining  are  to  clean  the 
cover-slips  thoroughly,  to  mix  the  culture  with  water  and  have  no 
culture  media  with  it,  to  fix  gently,  and  not  to  overheat  the  stain. 
Even  in  expert  practised  hands  it  is  not  always  easy  to  demon- 
strate flagella  readily. 


STAINING   DIPHTHERIA  BACILLI  10$ 


Neisser's  method  of  staining  the  diphtheria  bacillus. 
Two  stains  are  needed  (Fig.  21): 


FIG.  2i.— B.  Diphtheria  stained  by  Neisser's  method.     (Williams.) 


A    Methylene  blue 

i  gram. 

95  percent  alcohol  
Water 

20  c.c. 
o^o  c.c. 

Mix  and  add 
Glacial  acetic  acid 

50  c.c. 

B    Vesuvin 

2  grams. 

Distilled  water.  .  . 

.     1000  C.C. 

The  staining  steps  are  as  follows: 

1.  Prepare  film,  fix  and  dry. 

2.  Pour  on  "A"  for  thirty  seconds. 

3.  Wash  well  in  water. 

4.  Dry  and  pour  on  "B"  for  thirty  seconds. 

5.  Wash,  dry  and  mount. 

The  protoplasm  of  the  bacilli  will  be  stained  brown,  and  the 
characteristic  (diagnostic)  chromatin  points  will  be  stained  a 
deep  blue  black. 


106  STUDY   OF  BACTERIA 

Tubercle  Bacillus  Stain 

1.  Spread  the  sputum,  pus  or  culture,  over  the  surface  of  the  cover-slip. 
Allow  the  preparation  to  thoroughly  dry. 

2.  Fix  in  flame  and  cool. 

3.  Pour  carbol-fuchsin  over  the  slide  and  heat  with  steaming  for  five  min- 
utes.    Young  bacilli  in  tubercles  and  other  fluids  are  very  difficult  to  stain 
in  this  way.     The  preparation  containing  them  should  be  stood  in  cold  carbol- 
fuchsin  for  twenty-four  hours.     This  method  stains  everything  on  the  slide. 

4.  Wash  in  water. 

5.  Decolorize  the  preparation  with  a  25  percent  solution  of  sulphuric  acid 
in  water  until  the  red  color  is  lost.     Repeat  this  once  or  twice. 

6.  Wash  and  counterstain  with  Loffler's  methylene  blue. 

7.  Dry  and  mount. 

Gabbet's  solution,  methylene  blue  2  grams,  H2SO4  25  c.c., 
water  75  c.c.,  is  a  very  useful,  convenient  decolorizer  and  counter- 
stain  for  sputum. 

In  such  a  preparation,  if  tubercle  or  other  acid-fast  bacilli  are 
present,  the  bacilli  will  be  colored  a  brilliant  red,  while  the  pus 
cells,  epithelial  cells,  and  other  bacteria  will  be  stained  blue. 

The  microscope  dark  field  illumination  enables  one  to  see 
flagella  and  capsules.  This  illumination  is  obtained  by  blocking 
out  the  central  portion  of  the  Abbe  condenser  in  the  substage  of 
the  microscope.  Light  is  admitted  only  from  the  sides  and  objects 
in  the  field  at  the  point  of  crossing  of  the  rays  reflect  these  from 
their  sides.  India  ink  may  be  used  as  a  background  for  bacteria 
that  stain  poorly  and  have  low  refractive  index. 

Protozoa  are  stained  by  Wright's  or  Giemsa's  method  in  one  of 
its  various  forms.  Spirochetes,  particularly  that  of  syphilis, 
may  be  stained  by  Giemsa's  methocl  but  show  up  more  clearly 
in  the  following  technic  of  Stern. 

1.  Dry  film  in  incubator  for  several  hours. 

2.  Immerse  in   10   percent  aqueous  silver   nitrate  in  diffuse 
daylight,  six  hours  to  three  days,  depending  on  thickness  of  smear 
and  need  for  haste. 


TUBERCLE  BACILLUS   STAIN  107 

3.  Correct  color  for  smear  is  a  dull  gray  brown  with  metallic 
sheen — wash  in  water,  dry  and  examine.  Spirochetes  are  deep 
brown  or  black,  cells  delicate  brown. 

Microscopic  objects  are  measured  by  viewing  with  an  ocular 
fitted  with  a  graduated  glass  disc.  Their  values  are  indicated 
on  the  apparatus. 


CHAPTER  VI 
BACTERIOLOGICAL  LABORATORY  TECHNIC 

In  order  to  study  bacteria  by  other  methods  than  the  simple 
examination  of  their  morphology  by  means  of  stains,  and  by  the 
hang-drop,  or  block  method,  they  must  be  cultivated  either  in 
the  bodies  of  experiment  animals,  or  in  culture  media  artificially 
prepared.  The  latter  method  is  the  most  widely  used  in  labora- 
tories. It  is  necessary,  in  order  to  study  bacteria,  that  the  media 
shall  not  contain  any  extraneous  bacteria  to  begin  with,  and 
that  they  shall  be  cultivated  under  such  conditions  that  such 
bacteria  cannot  reach  the  media  at  any  time.  To  accomplish  all 
this,  the  culture  media  must  be  kept  in  glass  vessels,  such  as  test- 
tubes  and  flasks  that  have  been  sterilized.  And,  since  all  animal 
and  vegetable  substances,  not  actually  alive,  are  teeming  with 
a  multitude  of  bacteria,  these  substances  must  be  sterilized  too, 
in  order  that  the  media  shall  be  free  from  any  living  organisms. 

Glassware  is  cleaned  by  boiling  with  soap  suds  or  powder  or  if 
very  dirty  by  immersion  in  saturated  watery  solution  of  bichro- 
mate of  potash  plus  an  equal  part  of  sulphuric  acid.  This  latter 
must  be  very  carefully  washed  away  in  running  water. 

Glassware,  such  as  pipettes,  Petri  dishes,  flasks  and  test-tubes, 
are  sterilized  best  by  dry  heat  in  hot-air  sterilizers.  The  appara- 
tus is  subjected  to  a  temperature  of  i5o°C.  for  one  hour,  or  until 
the  cotton  plugs  are  slightly  brown.  The  glassware  should  be 
put  in  wire  baskets  and  the  test-tubes  should  be  kept  erect. 
Petri  dishes  are  best  sterilized  in  a  wrapping  of  paper.  Flasks 
and  test-tubes*  are  always  plugged  with  raw  cotton,  which  pre- 
vents the  ingress  of  bacteria,  while  air  can  reach  the  media 
through  it  freely. 

108 


STERILIZATION 


I09 


Sterilization  of  culture  media  is  accomplished  in  steam  steril- 
izers of  two  patterns;  of  these,  the  autoclave,  using  steam  under 
pressure,  is  the  most  satisfactory  and  is  most  generally  used  at 
present. 

The  baskets  containing  the  culture  media  are  placed  in  the 
autoclave  after  sufficient  water  has  been  put  in  it.  The  instru- 


FIG.  22. — Autoclave. 

ment  must  never  be  allowed  to  run  without  water.  The  lid  is 
screwed  down  and  the  flame  started;  free  flowing  steam  should 
escape  from  the  valve  before  the  latter  is  shut.  When  the 
pressure  has  risen  to  i  atmosphere  (15  pounds)  or  i2o°C.  and 
held  there  for  twenty  minutes,  all  bacteria  are  destroyed,  and 
the  media  can  be  safely  assumed  to  be  sterilized.  If  media 
containing  sugar  or  gelatine  are  to  be  sterilized,  the  temperature 
should  not  run  above  no°C.,  since,  if  this  is  done  the  gelatine  will 


no 


BACTERIOLOGICAL  LABORATORY  TECHNIC 


not  solidify  when  cold,  the  sugar  is  caramelized  and  the  media 
blackened. 

Potato  tubes  are  harder  to  sterilize  at  times,  and  it  is  safer 
to  repeat  the  operation  in  twenty-four  hours. 

Fractional  method  of  sterilization,  or  Tyndallization,  is  accom- 
plished by  heating  the  media  to  ioo°C.  on  three  successive  days 


FIG.  23. — Arnold  sterilizer. 

in  a  lyoch  or  Arnold  sterilizer.  By  heating  culture  media  to  this 
temperature,  all  the  vegetative,  or  adult,  forms  are  killed,  while 
the  spores  are  not  affected;  after  the  first  sterilization,  at  room 
temperature,  the  spores  vegetate  and  become  adult  bacteria, 
when  on  the  second  sterilization  they  are  non-resistant  to  ioo°C. 
and  are  killed.  Spores  remaining  after  this  develop  into  adult 
forms  again  and  are  killed  on  the  third  day,  at  the  third  steriliza- 
tion. This  fractional  sterilization  is  employed  under  many 


BACTERIA   CULTIVATION 


III 


circumstances,  and  is  certainly  the  best  for  media  containing 
carbohydrates  of  any  kind.  To  be  effective,  the  media  must  be 
exposed  to  a  temperature  of  ioo°C.  for  thirty  minutes,  that  is, 
thirty  minutes  after  the  steam  has  begun  to  form.  Overheating 
of  sugars  causes  them  to  caramelize  and  turn  black. 


FIG.  24. — Incubator. 

Bacteria  that  grow  best  at  a  temperature  of  37°C.  (most  of  the 
pathogenic  ones  do)  develop  more  rapidly  and  luxuriantly  in  an 
incubator,  or  thermostat.  Indeed  some  organisms,  like  the 
tubercle  bacillus,  cannot  be  cultivated  without  it.  An  incubator 
comprises  an  air  chamber  surrounded  by  a  water  chamber,  and 
this,  in  turn,  is  surrounded  by  another  air  chamber.  It  is  essen- 
tial that  the  interior  of  the  incubator  be  kept  at  an  even,  unvary- 
ing temperature.  This  is  accomplished  by  using  a  small  bunsen 
flame  under  the  incubator.  The  heat  from  the  flame  warms  the 
outer  air  chamber  or  jacket,  and  it  in  turn  warms  the  water 


112         BACTERIOLOGICAL  LABORATORY  TECHNIC 

jacket,  and  the  interior  air  chamber,  where  the  cultures  are  kept, 
is  thus  heated  to  the  required  temperature.  The  amount  of  heat 
is  automatically  regulated  by  a  thermo-regulator,  which  dimin- 
ishes the  gas  supply  if  the  temperature  runs  too  high,  or  increases 
it  if  it  runs  too  low.  The  Roux  regulator  is  the  simplest  and  most 
efficient  one. 


FIG.  25. — Blood  serum  coagulating  apparatus. 

A  serum  coagulating  apparatus  is  needed  in  laboratories  in 
order  to  coagulate  the  tubes  of  blood  serum  (Fig.  25). 

Serum  tubes  are  coagulated  in  it  at  a  temperature  of  about  7o°C. 
They  are  then  sterilized  by  heating  them  either  by  the  fractional 
method  or  in  the  autoclave. 

The  separation  of  bacteria  from  the  bouillon  in  which  they 
grow  for  the  preparation  of  toxins  requires  the  use  of  a  bacteria 
or  germ  proof  filter,  the  best  type  of  which  is  the  Chamberland  or 
Pasteur  unglazed  porcelain  filter.  These  filters  are  of  varying 
grades  of  fineness,  and  are  so  made  as  to  be  easily  sterilized.  The 
common  pathogenic  bacteria  cannot  pass  through  the  pores  of 
the  ordinary  filter,  but  toxic  agents  are  known  to  pass  through  the 
finest  filters,  though  they  cannot  be  discovered,  as  they  are 
submicroscopic. 

To  operate  the  porcelain  filter  it  must  fit  into  the  neck  of  a 


NUTRIENT   MEDIA 


vessel  very  tightly,  so  that  a  vacuum  may  be  maintained  in  the 
latter  by  means  of  an  air  pump. 

Collodion  sacs  are  sometimes  used  in  animal  experiments. 
Bouillon  cultures  are  placed  within  the 
sacs,  which  are  then  inserted  in  the  ab- 
domen of  an  animal  and  left  there.  The 
sac  is  made  of  coUodion  because  it  is 
non-absorbent  and  allows  the  bacterial 
juices  and  products  to  osmose  outward 
and  be  absorbed  by  the  animal,  while  the 
animal  fluids  percolate  into  the  sac. 
There  are  several  very  ingenious  ways  of 
making  these  sacs,  but  the  details  are  too 
elaborate  to  be  described  here. 

BOUILLON 

Bouillon  or  broth  is  the  most  useful  of 
all  the  nutrient  media,  since  it  is  not  only 
used  as  a  liquid  medium,  but  by  the  addi- 
tion of  gelatine,  or  agar,  it  is  converted 
into  solid  media. 

There  are  two  methods  of  making 
bouillon: 

Method  i. 

Take  500  grams  of  lean  beef  free  from  all  fat,  chop  it  fine  and 
cover  with  1,000  c.c.  of  water,  shake  and  place  on  the  ice  over- 
night. Then  squeeze  the  fluid  out  of  the  met  by  means  of  a 
cloth,  and  supply  enough  water  to  make  a  litre.  Inoculate  this 
meat  juice  with  a  fluid  culture  of  the  colon  bacillus  for  the  pur- 
pose of  fermenting  the  meat  sugar.  For  this  purpose  the  inocu- 
lated juice  is  allowed  to  stand  at  room  temperature  overnight. 
Bring  to  a  boil  and  add 

10  grams  of  Witte's  peptone. 
5  grams  common  salt. 


FIG.  26. — Kitasato 
filter  for  filtering  toxins. 
(Williams.) 


114        BACTERIOLOGICAL  LABORATORY  TECHNIC 

Weight  the  saucepan  and  contents  and  heat  to  6o°C.  Supply 
the  water  lost  by  evaporation.  Neutralize  either  by  adding 
sufficient  sodium  hydrate,  10  percent  solution,  until  red  litmus 
paper  is  colored  a  faint  blue,  or  else  titrate  10  c.c.  of  the  mixture 
with  a  decinormal  solution  of  sodium  hydrate,  using  phenol- 
phthalein  as  an  indicator,  and  after  finding  how  much  of  a  normal 
solution  is  required  to  neutralize  990  c.c.  (1,000  c.c. — 10  c.c. 
used  for  titration)  this  normal  solution  is  added.  The  mixture 
thus  neutralized  is  then  boiled  for  five  minutes  and  the  weight 
restored.  After  boiling,  from  .5  percent  to  1.5  percent  normal 
hydrochloric  acid  solution  is  added  and  the  acidity  thus  produced 
is  spoken  of  as  +  .5  percent  or  +  1.5  percent  as  the  case  may  be. 
Upon  boiling,  the  albumins  are  coagulated  by  heat,  and  the 
phosphates  are  thrown  down.  The  acid  re-dissolves  the  latter. 
The  former  must  be  removed  by  filtration.  The  filtrate  is  a  clear 
straw-colored  fluid  of  an  acid  reaction  which  should  not  become 
cloudy  upon  boiling.  This  is  then  run  into  flasks  or  test-tubes 
and  sterilized. 

The  second  method  is  much  more  convenient,  and  is  prepared 
by  adding  3  grams  of  Liebig's  beef  extract  to  a  litre  of  water,  and 
adding  the  peptone  and  salt,  as  in  the  previous  method,  and  pro- 
ceeding as  before.  To  filter  the  bouillon,  the  filter  paper  must 
be  folded  many  times,  and  the  funnel  must  be  carefully  cleaned. 

Newer  methods  for  the  titration  of  media  have  been  constructed 
on  a  physico-chemical  basis,  attempt  being  made  to  estimate 
exact  reaction  in  terms  of  ionic  dissociation  and  hydrogen  concen- 
tration. Distilled  water  contains  ionizable  hydrogen  according 
to  the  following  formula  i  +  10  —  7  =  log.  —  7,  a  mathematical 
statement  abbreviated  for  convenience  to  7.,  or  the  symbol  of 
strict  neutrality  and  called  the  Ph  or  hydrogen  concentration. 
As  the  ionization  of  hydrogen  increases,  acidity  becomes  greater 
and  the  numerical  factor  drops  to  6.8  or  lower;  as  the  hydrogen 
value  decreases  the  figure  rises.  Substances  in  solution,  such  as 
salts  or  organic  matter  act  as  buffers  or  agents  which  attempt  to 


NUTRIENT   MEDIA 


keep  the  hydrogen  ion  concentration  fixed.  Dilution  does  not 
change  the  concentration.  Mixtures  of  acid  and  alkaline  salts 
which  will  give  hydrogen  ion  concentration  ranging  from  Ph 
values  of  5.  (acid)  to  9.  (alkali)  have  been  prepared  for  use  in 
bacteriology.  To  these  solutions  may  be  added  dye  indicators. 
Different  dyes  have  different  ranges  of  color  changes.  The  most 
useful  are  the  following: 


Range]  of  rPh) 
5.2  —  6.8 
6.0  —  7.6 
6.8  —  8.4 


Brom  cresol  purple          yellow-purple 

Brom  thymol  blue  yellow-blue 

Phenol  red  yellow-red 

Litmus  which  has  so  long  served  a  useful  purpose  in  the  laboratory 

has  a  Ph  of  about  6.8.     A  convenient  method  for  practical  use  in 

the  laboratory  is  as  follows : 

Materials : 

1.  Chemically  clean  test  tubes. 

2.  Freshly  distilled  water. 

3.  N/i5  KH2PO4  (primary  phosphate)  solution.     (1.078  gm.  to  litre  of 
water.) 

4.  N/i5  Na2HPO4  (secondary  phosphate)  solution  (11.996  gm.  per  litre 
of  water.) 

5.  Phenolsulphonphthalein  .01  percent  water  solution  (or  phenol  red). 

6.  N/20  NaOH,  N  NaOH. 

7.  N/20  HC1,  N  HC1. 

Phosphate  mixture  for  Ph  values  7.0  to  8  o. 


Ph  value 


Amt.  of  primary 

phos.  in  c.c. 

19.4 

14.2 

9-7 
6-5 
4-3 
2-5 
Procedure. 

Add  20  c.c.  freshly  distilled  water  to  chemically  clean  test  tube.  Add 
10  drops  phenol  red  and  5  c.c.  of  medium  to  be  titrated.  Compare  with 
standard. 


7.0  - 


7-8 
8.0 


Amt.  of  secondary  phos. 

in  c.c. 

Quantities  to  be 
mixed  to  get  in- 
dicated Ph  value. 
5  drops  indicator 
to  each. 


30.6 
35-8 
40.3 
43-5 
45-7 
47-5 


Il6         BACTERIOLOGICAL  LABORATORY  TECHNIC 

Titrate  with  N/20  NaOH  or  N/2o  HC1  to  match  tint  of  stand- 
ard tube  corresponding  to  the  Ph  desired.  Calculate  the  amount 
of  normal  alkali  or  acid  to  be  added  to  the  medium  to  give  the 
proper  reaction. 

If  the  natural  color  of  the  medium  makes  it  difficult  to  match 
with  the  standard  solutions,  a  tube  of  20  percent  solution  of  the 
medium  in  water  may  be  placed  back  of  the  standard  solution. 
The  best  generally  useful  reaction  is  7.4,  a  figure  suitable  for 
most  pathogenic  organisms. 


GELATINE 

To  make  gelatine,  bouillon  is  made  to  which  gelatine  is  added 
in  order  to  render  it  solid.    The  following  steps  are  taken: 

(a)  Take  a  litre  of  water  in  a  saucepan  and  add  chopped  beef  or 
beef   extract   as   in   bouillon.     After   standing   overnight 
squeeze  the  beef  and  extract  the  juice. 

(b)  Add  i  percent  peptone,  .5  percent  salt,  10  percent  to  15 
percent  best  gelatine  and  weigh. 

(c)  Heat  until  ingredients  are  all  dissolved. 

(d)  Neutralize,  gelatine  is  highly  acid  and  requires  much  alkali. 

(e)  Boil  five  minutes  and  restore  weight,  boil  till  albumin 
coagulates. 

(/)  Cool  to  6o°C.  and  add  an  egg  well  beaten  up  in  water. 
(g)  Boil  slowly  till  all  the  egg  is  coagulated.     This  clears  the 

jnedium  of  fine  particles  that  are  not  removed  by  filtration. 

Add  .5  percent  normal  hydrochloric  acid. 
(h)  Filter  through  absorbent  cotton  on  a  funnel  previously  wet 

with  boiling  water. 
(i)  Tube  and  sterilize  in  autoclave  for  fifteen  minutes  at  i  io°C. 

Litmus,  or  lacmoid,  or  neutral  red  may  be  added  to  the 

gelatine  as  an  indicator. 


AGAR-AGAR  117 

AGAR-AGAR 

To  make  agar : 

(a)  Take  20  grams  of  powdered  or  chopped  agar. 

(b)  Add  to  500  c.c.  of  water,  place  in  a  can  in  autoclave  and 
heat  to  1 2o°C.    Then  cool. 

(c)  Add  this  to  500  c.c.  of  bouillon  of  double  strength,  making 
1,000  c.c. 

(d)  Neutralize. 

(e)  Cool  to  6o°C. 

(/)  Add  the  egg  to  the  mixture,  stir. 

(g)  Boil  till  egg  is  coagulated  thoroughly. 

(h)  Titrate  and  adjust  to  desired  acidity  as  given  under  bouil- 
lon, and  while  boiling  hot,  filter  through  absorbent  cotton 
wet  with  boiling  water. 

(i)  Run  into  tubes.  Sterilize.  Slope  the  tubes  for  twelve 
hours  and  store  in  dark  place. 

To  make  glycerine  agar  add  5  percent  of  glycerine  to  the  agar 
before  neutralizing.  To  make  agar  for  tubercle  bacilli,  veal 
bouillon  may  be  employed,  and  glycerine  must  be  added. 

Litmus  Milk 

Carefully  skimmed  milk,  to  which  litmus  tincture  has  been 
added,  is  run  into  tubes  and  sterilized.  This  is  a  valuable  culture 
medium.  It  is  also  a  reagent. 

Potato  Tubes 

i.  Wash  some  large  potatoes  and  with  a  Ravenel  potato  cutter, 
cut  out  semi-cylinders  of  potato.  Immerse  in  running  water  over- 
night, in  order  to  prevent  them  from  turning  black.  It  is  well 
to  wash  these  bits  of  potato  with  i- 10,000  bichloride  of  mercury 
six  hours  and  running  water  over  night.  Some  laboratories  soak 
their  slices  in  sodium  carbonate  solution.  It  is  desirable  to  know 


n8 


BACTERIOLOGICAL  LABORATORY  TECHNIC 


the  reaction  of  the  medium  and  each  batch  should  be  tested,  then 
marked  whether  faintly  or  strongly  acid  or  alkaline. 

Thrust  absorbent  cotton  to  the  bottom  of  the  tube  and  wet 
with  distilled  water;  place  the  potato  upon  the 
cotton,  then  plug  the  tube  and  sterilize  in  auto- 
clave twice.  The  tubes  should  be  sealed. 

PEPTONE  SOLUTION— Dunham 

Take  Peptone 10  grams. 

Salt 5  grams. 

Water 1,000  c.c. 

Mix.     Boil.     Filter  and  store  in  tubes  and  sterilize. 

This  is  used  to  demonstrate  the  production  of 
indol.     Reaction  should  be  neutral. 

SUGAR  MEDIA 

One  of  the  most  important  parts  of  determinative 
bacteriology  is  the  discovery  of  the  different  fer- 
mentative powers  upon  carbohydrates  of  otherwise 
similar  germs.  Monosaccharides  (dextrose  and 
galactose),  disaccharides  (lactose,  saccharose), 
alcohols  (glycerine  mannite)  and  some  starches 
(dextrin,  inulin)  are  in  common  use  daily  in  the 
laboratory  to  show  the  enzyme  action  of  various 
species,  indicated  by  acidity  and  gas  production. 
One  percent  solutions  of  these  carbohydrates  are  made  in  neutral 
broth  or  agar.  The  best  method  is  to  prepare  a  20  percent 
solution  of  the  material,  sterilize  it  in  the  steam  sterilizer  and  add 
from  this  to  the  stock  medium  sufficient  to  make  the  required 
percentage.  This  avoids  repeated  heating.  The  media  should 
be  neutral.  Addition  of  litmus  tincture  or  Andrade's  indicator 
(100  c.c.  of  .5  percent  watery  acid  fuchsin  decolorized  by  the 
addition  of  16  c.c.  N/i  NOOH)  will  supply  an  index  for  acid 
production. 


FIG.  27.— Po- 
tato in  culture 
tube.  (Wil- 
liams.) 


LOFFLER'S  BLOOD  SERUM  MIXTURE  119 

BLOOD  AGAR 

Is  prepared  by  adding  to  melted  agar  sterile  defibrinated  blood 
of  any  animal  in  the  proportion  of  i  of  blood  to  5  of  agar.  The 
mixture  may  then  be  allowed  to  harden  in  a  slanting  position  or 
poured  into  plates. 

BLOOD  SERUM 

The  blood  of  a  dog,  sheep  or  cow  drawn  under  strictly  aseptic 
precautions  is  collected  in  a  sterile  jar  and  after  the  serum  has 
separated,  it  is  run  into  tubes  by  sterile  pipettes  and  simply 
coagulated  by  heat.  Sterilization  is  not  necessary,  and  is  harm- 
ful for  the  growth  of  the  tubercle  bacilli,  because  salts  are  formed 
which  interfere  with  the  growth  of  the  bacteria.  If  this  serum 
be  mixed  with  3  or  4  parts  of  distilled  water  and  sterilized  five 
days  in  the  Arnold  at  7o°C.  it  will  be  a  slightly  turbid,  opalescent 
liquid  very  suitable  for  many  organisms,  particularly  cocci. 
This  medium  with  the  addition  of  bits  of  animal  tissue  is  a  good 
medium  for  spirochaetes. 

LOFFLER'S  BLOOD  SERUM  MIXTURE 

Blood  serum  of  an  ox,  a  sheep  or  a  horse  is  employed,  mixed 
with  bouillon  containing  i  percent  of  grape  sugar. 

Seventy-five  percent  of  blood  serum  is  mixed  with  25  percent 
bouillon.  This  is  run  into  sterilized  tubes  and  the  latter  are 
placed  in  a  blood  serum  coagulator  and  coagulated  in  a  sloping 
position  at  a  temperature  of  65°C.  or  thereabouts. 

After  they  are  coagulated  they  are  sterilized  by  heating  an 
hour  each  day  at  65°C.  five  successive  days,  or  at  95°C.  for  an 
hour  on  three  successive  days.  After  sterilization  the  tubes 
should  be  sealed  carefully. 

Ascitic  and  hydrocele  fluids  may  be  used  for  this  medium  or 
in  the  liquid  form  combined  with  plain  broth;  they  need  no 
addition  of  sugar  as  they  contain  a  small  percentage. 


120         BACTERIOLOGICAL  LABORATORY  TECHNIC 

SPECIAL  MEDIA 

Endo  medium  for  the  Typhoid  series.  Add  i  percent  lactose  to 
plain  neutral  agar  and  i  percent  of  the  following  solution:  10  c.c. 
of  10  percent  watery  sodium  sulphite  and  i  c.c.  of  saturated 
alcoholic  solution  of  fuchsin,  heated  in  the  Arnold  twenty  minutes. 
This  should  be  mixed  freshly  each  time  since  the  medium  is 
properly  colorless  but  becomes  a  pale  pink  on  standing,  being 
then  useless.  It  is  poured  into  plates  and  allowed  to  cool  and 
harden  thoroughly  before  use. 

Russell's  Medium. — Plain  neutral  agar  receives  i  percent 
lactose,  .1  percent  glucose  and  an  indicator,  either  litmus  or 
Andrade.  It  is  used  as  slants  with  a  deep  butt. 

The  first  medium  is  to  distinguish  typhoid  colonies  from  colon 
bacillus;  pale  bluish  round  colonies  versus  irregular  red  ones. 
The  Russell  medium  gives  with  typhoid,  a  colorless  surface 
growth  and  acid  in  the  butt;  paratyphoids  give  bubbles  of  fermen- 
tation in  the  butt;  colon  makes  distinct  red  slant  growth  and 
much  gas. 

Eggs  are  employed  as  culture  media.  The  yolks  and  whites  of 
a  number  of  eggs  are  shaken  together  in  a  flask  and  then  strained 
through  a  towel  to  remove  the  froth.  The  mixture  is  then  run 
into  tubes  and  coagulated  and  sterilized  like  blood  serum.  On 
this  mixture  the  tubercle  bacillus  grows  very  well. 

These  are  the  common  culture  media  used  in  laboratories.  For 
a  more  technical  description  of  the  manufacture  of  these  and 
other  media,  the  student  is  referred  to  books  devoted  to  labora- 
tory technique. 

Litmus  tincture  is  made  by  adding  a  large  handful  of  litmus 
cubes  to  a  pint  of  water  and  boiling  down  to  one-fourth  its  volume. 
This  is  then  filtered  through  paper  and  stored  after  sterilization. 

The  Study  of  the  Growth  of  Bacteria— Cultures 

Bacteria  growing  in  groups  on  culture  media  are  spoken  of  as 
colonies.  Aerobic  bacteria  may  be  made  to  grow  on  culture 


THE    STUDY   OF    THE    GROWTH    OF  BACTERIA  121 

media  by  simply  inoculating  the  media  with  some  pus  or  blood 
containing  them,  by  means  of  a  sterile  pipette  or  platinum  needle. 
But  such  cultures  are  made  up  of  colonies  of  different  sorts  of 
bacteria — some  pathogenic,  some  non-pathogenic,  etc.  To  sepa- 
rate the  various  bacteria  so  that  they  will  grow  in  isolated  groups, 
is  a  comparatively  easy  matter,  and  is  accomplished  in  several 
ways.  The  simplest  is  to  employ  several  tubes  of  agar  or  blood 


FIG.  28. — Colonies  in  gelatine  plate  showing  how  they  may  be  separated  and 
the  organisms  isolated.     (Williams.,) 

serum.  Over  the  surface  of  each  of  these,  a  platinum  loop  con- 
taining pus,  or  other  matter,  is  rubbed  successively.  These  tubes 
are  then  incubated.  After  a  few  hours,  the  first  one  exhibits  a 
copious  growth  of  many  different  kinds  of  bacteria  growing  con- 
fluently  together,  from  which  it  is  impossible  to  isolate  any  pure 
cultures.  The  second  tube  is  less  covered  with  bacteria  while, 
the  third,  instead  of  containing  a  mass  of  bacteria,  exhibits  tiny 
little  dots,  or  colonies  (pure  cultures)  growing  discretely  isolated. 
By  means  of  a  sterilized  platinum  needle  these  little  colonies 


122         BACTERIOLOGICAL  LABORATORY  TECHNIC 

may  be  fished  out  and  transplanted  to  fresh  culture  tubes,  and 
after  a  few  hours'  growth  they  become  pure  cultures.  An  old 
method  employed  in  many  laboratories,  in  breweries  and  orig- 
inated by  Pasteur  was  what  is  known  as  the  dilution  method. 


FIG.  29. — Series  of  stab  cultures  in  gelatine,  showing  modes  of  growth  of 
different  species  of  bacteria.     (Abbott.) 

Numerous  flasks  are  inoculated  by  matter  containing  bacteria 
very  highly  diluted  in  bouillon  and  by  means  of  a  sterile  pipette 
drops  of  this  highly  attenuated  mixture  are  dropped  into  flasks 
of  sterilized  bouillon  or  wort.  Most  of  the  flasks  will  show  a 
mixed  growth  but  a  few  will  show  only  one  kind  of  organism. 


THE   STUDY   OF   THE   GROWTH   OF  BACTERIA 


123 


Another  method  is  to  inject  some  matter  containing  patho- 
genic bacteria  into  a  rabbit  of  guinea  pig.  The  various  juices 
and  the  leucocytes  of  the  animal  destroy  the  non-pathogenic 
bacteria  and  a  pure  culture,  of  a  pathogenic  form,  may  be  isolated 
from  the  blood  or  pathological  lesions  at 
autopsy  and  transferred  to  culture  media. 

By  far  the  most  useful  and  ingenious 
method  of  procedure  is  the  Koch,  or  plate 
method.  Koch  was  the  first  to  employ  solid 
culture  media  for  this  purpose,  and  his 
method  depends  upon  the  principle  that  a 
liquid  culture  media  may  be  inoculated  with 
bacteria  and  then  spread  out  on  sterile  glass 
plates  or  dishes  where  it  quickly  hardens,  the 
bacteria  being  uniformly  separated  from  each 
other,  and  for  a  time  at  least  kept  isolated 
by  means  of  the  solid  media,  and  after  they 
have  developed  into  isolated  colonies  they 
may  be  transplanted  to  tubes  of  media  in 
which  they  may  be  stored.  In  another  way 
if  a  man  wanted  to  secure  a  pure  lot  of  seed 
of  a  single  variety  from  a  multitude  of  many 
kinds,  it  would  perhaps  be  impossible  to 
pick  out  by  hand  the  seed  wanted  because 
of  their  fewness  and  smallness,  but  if  he 
sowed  them  and  waited  until  the  plants 
developed  they  could  then  be  identified  and  gathered  (Abbott). 
Thus  it  is  with  plate  cultures. 

To  isolate  a  pure  culture  of  bacteria,  say  the  Bacillus  pyocya- 
neus  from  pus,  the  following  procedure  is  adopted  in  this  method. 

Three  sterilized  petri  dishes,  and  three  tubes  of  agar  or  gelatine 
melted  at  4o°C.  are  used.  A  loopful  of  pus  is  taken  up  by  a  ster- 
ilized platinum  loop  and  mixed  with  the  gelatine  of  the  first  tube. 
To  do  this  the  tube  is  held  across  the  left  hand  in  a  horizontal 


FIG.  30. — Needles 
used  for  inoculating 
media. 


124         BACTERIOLOGICAL  LABORATORY  TECHNIC 

position  and  the  cotton  plug  is  removed,  and  held  by  its  outside 
end  between  the  fingers  of  the  left  hand,  care  being  taken  to  pre- 
vent the  tubal  part  of  the  plug  touching  anything  and  being  con- 
taminated. The  platinum  loop  is  then  slowly  and  carefully 
introduced  into  the  median,  and  stirred  around  so  that  the  tube 
walls  are  not  touched.  The  needle  is  again  sterilized  and  tube 
number  two  is  held  in  the  palm  of  the  left  hand  parallel  to 
the  first  one  and  its  plug  is  removed  also;  then  with  a  carefully 
sterilized  needle,  three  loops  of  the  inoculated  gelatine  are  re- 
moved from  number  one  and  mixed  with  number  two  tube. 


FIG.  31. — Method  of  inoculating  culture  media.     (Williams.)  ' 

The  needle  is  then  again  carefully  sterilized  in  the  flame,  the  plug 
of  number  one  is  carefully  replaced  and  another  tube,  number 
three,  is  held  in  the  palm  of  the  left  hand  and  its  plug  is  carefully 
removed  and  held  as  the  previous  ones  were.  With  the  sterilized 
loop  three  loopfuls  of  the  gelatine  from  number  two  are  carefully 
introduced  into  number  three  and  the  needle  is  then  sterilized 
and  p'ut  aside.  The  petri  dishes  should  now  be  laid  on  a  cold  level 
slab,  and  the  contents  of  the  tubes  run  into  the  different  dishes. 
Tube  number  one  is  taken  first;  the  lip  of  the  tube  is  wiped  with 
the  cotton  plug  and  then  held  in  the  flame  to  destroy  all  bacteria 
clinging  to  it.  The  lid  of  a  petri  dish  is  carefully  and  partially 
lifted  and  the  contents  of  the  tube 'rapidly  and  evenly  poured 
over  the  bottom  of  the  plate,  and  the  lid  quickly  replaced. 


THE   STUDY   OF   THE   GROWTH   OF  BACTERIA  125 


FIG.  32. — Dilution  method  of  making  cultures,  i,  Is  first  tube  containing 
great  number  of  colonies;  2,  contains  less  number;  3,  relatively  few. 
(Williams.) 


126         BACTERIOLOGICAL  LABORATORY  TECHNIC 

This  procedure  is  followed  with  the  other  tubes,  and  then  the 
plates  or  dishes  are  put  in  a  cool  dark  place,  and  the  tubes  are  put 
into  a  solution  of  bichloride  of  mercury,  or  into  boiling  water. 

The  plates  should  be  examined  from  time  to  time.  After  several 
days  a  perfect  cloud  of  round  colonies  are  seen  in  number  one;  a 
large  number  in  No.  2  and  a  much  fewer  number,  say  fifty,  in  No.  3. 
It  is  an  easy  matter  then  to  pick  out  a  colony  that  is  surrounded 
by  a  bluish-green  halo  and  transfer  it  to  a  tube  of  agar  or  bouillon. 
In  the  case  of  pus  it  is  more  than  probable  that  the  colony  is  that 
of  the  pyocyaneus  bacillus,  and  that  it  contains  nothing  but  these 
bacilli.  It  must  be  studied  in  a  dozen  ways,  before  it  is  certain 
that  it  is  this  bacillus,  but  the  preceding  method  is  a  necessary 
primary  step  to  secure  this  organism  in  pure  culture  and  may 
be  taken  as  a  pattern  for  all  plate  methods. 

Agar  plates  are  usually  used  since  they  have  this  advantage — 
they  do  not  melt  at  37°C.  incubator  temperature.  When  agar  is 
used  it  must  be  melted  at  ioo°C.  and  cooled  below  48°C.  and 
above  43°C.  Above  48°C.  bacteria  may  be  killed.  Below  43°C. 
the  agar  begins  to  harden,  so  this  method  must  be  performed 
quickly;  the  plates  should  be  slightly  warmed,  the  culture  poured 
on  and  the  agar  hardened,  they  must  be  invented  in  the  incubator, 
since  the  water  of  condensation  forming  in  the  lids  of  the  plates 
often  falls  and  washes  one  colony  into  another.  When  gelatine 
plates  are  made,  they  must  be  kept  at  2o°-25°C.  It  is  often  of 
advantage  to  cool  the  plates  by  means  of  ice,  before  they  are 
filled. 

The  so-called  "Stroke  plates"  are  extremely  useful  for  hospital 
bacteriology.  The  agar  is  softened,  poured  into  plates,  allowed  to 
harden  and  the  material  to  be  examined  is  smeared  upon  the  firm 
surface  by  a  flattened  platinum  rod,  a  "Spatula."  The  separate 
colonies  develop  along  the  lines  of  spread  and  can  be  isolated  to 
individual  tubes  as  given  above. 

Roll  Citllure. — Instead  of  pouring  out  the  contents  of  the  in- 
oculated tubes  the  gelatine  may  be  made  to  harden  on  the  walls  of 


ROLL   CULTURE  127 

the  tubes  by  quickly  rotating  the  tube  in  a  groove  melted  in  a 
block  of  ice.  The  centrifugal  force  distributes  the  gelatine  over 
the  glass,  and  the  ice  hardens  it  rapidly  while  in  contact  with  the 
glass.  Such  tubes  are  veritable  plates,  and  in  them  colonies  of 
bacteria  often  grow  as  well  as  on  the  plates  and  may  be  fished 
out. 

The  various  characteristics  of  bacterial  growth  may  be  studied 
in  cultures.  Some  organismal  cultures  grow  rapidly  and  luxuri- 
antly; some  discretely  and  slowly;  colors  and  odors  are  produced 
by  some;  gelatine  is  liquefied  by  many,  while  others  do  not  liquefy 
gelatine.  Milk  is  curdled  and  digested  by  some;  gas  and  acids 
produced  by  others.  These  various  characteristics  enable  us  to 
identify  and  differentiate  bacteria. 

The  cultivation  of  bacteria  in  the  laboratory  has  for  its  purpose 
a  demonstration  of  their  vital  activities.  This  may  indicate  only 
their  botanical  character  or  it  may  show  their  relation  to  disease. 
In  order  that  we  may  classify  germs  systematically  certain  criteria 
have  been  established  which  when  added  together  permit  us  to 
identify  and  name  the  organisms.  This  is  called  determinative 
bacteriology.  The  principal  characters  to  be  noted  are  com- 
plete morphology,  staining  characters,  particularly  with  Gram's 
method,  colonial  growth  on  agar  and  gelatine,  potato,  blood 
serum,  milk,  sometimes  inorganic  salt  solutions,  the  enzymic 
products  as  indicated  by  fermentation  of  carbohydrates  and 
solution  of  proteins  like  milk  curd  and  gelatine.  With  this  last 
comes  ammonia  and  nitrite  productions.  The  optimum  tempera- 
ture and  media,  and  resistance  to  physical  and  chemical  agencies 
must  be  taken  into  consideration.  For  pathogenic  bacteria  we 
establish  as  far  as  possible  the  relations  with  lower  animals. 
This  includes,  of  course,  the  production  of  soluble  toxins  and 
endotoxins. 

The  chemical  activities  of  many  bacteria  are  well  displayed  in 
litmus  milk  culture  in  which  decolorization,  acid  or  alkali  forma- 
tion, coagulation  and  clot  digestion  are  the  important  ones. 


128         BACTERIOLOGICAL  LABORATORY  TECHNIC 

The  property  of  converting  sugar  into  acids  and  gases  is  best 
studied  in  fermentation  tubes. 

Into  sterile  fermentation  tubes  bouillon  containing  sugar  is  run, 
these  are  plugged  and  sterilized.  They  may  be  inoculated  with 
bacteria  and  if  gas  production  occurs  it  is  quickly  manifested  in 
the  closed  arm.  The  component  gases  may  be  studied  and  the 
various  properties  determined.  This  gas  ratio  is  of  use  in  identi- 


FIG.  33. — Fermentation  tube.     (Williams.) 

fying  various  bacteria  and  differentiating  them.  The  closed  arm 
of  the  tube  being  shut  off  from  free  air  by  the  amount  of  bouillon 
in  the  open  arm  is  practically  an  anaerobic  tube  and  is  employed 
for  this  purpose.  Bacteria  that  grow  only  in  the  closed  arm  are 
considered  anaerobes.  By  inoculating  a  gelatine  tube  with  bac- 
teria while  it  is  melted  and  then  letting  it  solidify,  previously 
shaking  the  tube  vigorously,  gas  formation  will  be  speedily  mani- 


FERMENTATION   METHODS  1 29 

fested  by  the  presence  of  bubbles.  Acids  are  detected  in  cultures 
by  the  employment  of  various  indicators  in  the  culture  media. 
Litmus,  lacmoid,  andrade,  and  neutral  red  are  used  for  this  pur- 
pose. By  titrating  bouillon  of  previous  known  acidity  with  a 
decinormal  soda  solution,  the  amount  of  acid  produced  by  differ- 
ent bacteria  can  be  estimated. 

Various  sugars  are  fermented  by  bacteria,  and  lactic,  acetic, 
and  butyric  acids  are  produced.  Indol  is  also  produced  by  many 
bacteria  (colon  bacillus,  cholera  bacillus),  and  its  presence  in  cul- 
ture is  an  important  means  of  identifying  different  bacteria.  The 
organism  to  be  studied  must  be  grown  in  culture  media  known  to 
be  free  from  indol.  For  this  purpose,  all  meat  extracts  must  be 
excluded  and  a  simple  solution  of  peptone  and  salt,  run  into  tubes 
and  sterilized,  is  used.  After  bacteria  have  grown  in  this  media 
for  several  days  the  indol  produced,  if  it  is  produced,  is  detected 
by  adding  a  few  drops  of  pure  sulphuric  acid.  If  a  red  color 
(nitroso-indol)  is  not  produced,  a  few  drops  of  sodium  nitrite  solu- 
tion (.02  gram  to  100  c.c.  of  water)  must  be  added,  and  if  a  pink 
to  deep  red  color  does  appear  it  may  be  safely  assumed  that  indol 
is  present. 

Ammonia  is  detected  in  culture  by  suspending  a  piece  of  paper 
wet  with  Nessler's  reagent  above  a  bouillon  culture  of  a  given  or- 
ganism. If  a  yellow  to  brown  color  is  produced  ammonia  is 
present. 

Nitrites  are  detected  by  growing  the  organism  in  a  solution  of  a 
nitrate  (.02  gram  potassium  nitrate,  10  grams  peptone  in  1,000 
c.c.  of  water). 

Incubate  for  a  week  and  then  add  i  c.c.  each  of  the  following 
solutions : 

. 

(a)  Sulphuric  acid .5  gram. 

Acetic  acid 150      c.c. 

(6)  Amido  naphthaline .  i  gram. 

Water . 20    c.c. 

Boil,  filter,  and  add  180  c.c.  of  dilute  acetic  acid. 
9 


130 


BACTERIOLOGICAL  LABORATORY  TECHNIC 


If  nitrites  are  present  a  pink  color  is  produced  by  these  reagents. 
Enzymes  may  be  detected  by  noting  whether  gelatine  is  liquefied, 
or  milk  curd  digested.  Both  these  actions  are  evidences  of  the 
presence  of  enzymes. 

Bacteria  growing  exclusively  in  the  absence  of  oxygen  are 
known  as  anaerobes;  to  cultivate  these  successively  various  forms 
of  apparatus  are  necessary. 


FIG.  34. — A  streak  made  in  agar  by  a  needle  inoculated  with  aerobic  bacilli 
and  then  covered  at  one  spot  with  cover-glass.  The  aerobic  organisms  will 
not  grow  in  the  anaerobic  conditions  under  the  glass.  (Williams.; 

The  following  methods  are  pursued  in  ordinary  laboratory 
manipulations : 

1.  Exclusion  of  oxygen. 

2.  Exhaustion  of  oxygen  by  means  of  an  air-pump. 

3.  Absorption    of  oxygen  by  means  of  chemicals  that  absorb 
oxygen  from  the  air.     A  mixture  of  pyrogallic  acid  and  sodium 
hydrate  absorbs  oxygen  rapidly,  leaving  nitrogen  only  in  the 
chamber. 


ANAEROBIC   METHODS  131 

4.  Displacement  of  air  by  means  of  an  air-pump  and  allowing 
hydrogen  to  enter  the  vacuum. 

Under  \hzfirst  method  we  may  either  exclude  oxygen  by  laying 
sheets  of  sterile  mica  or  a  cover-glass  on  the  surface  of  the  agar 
or  gelatine  plates  (Fig.  34),  thus  excluding  air,  or  deep  punc- 
tures may  be  made  in  tubes  half  filled  with  gelatine  or  agar,  for 
growths  often  occur  in  the  depths  of  the  medium,  especially  if 


FIG.  35.— Novyjar. 


the  latter  has  been  boiled  previously  to  expel  the  oxygen;  or, 
instead  of  mica,  sterile  paraffine  may  be  poured  over  the  top  of 
the  tube.  The  layer  of  paraffine  excludes  the  air.  Flasks  filled 
with  bouillon,  or  tubes  filled  with  bouillon,  or  melted  agar  may  be 
inoculated  with  an  anaerobic  culture,  but  the  filling  of  the  vessel 
with  the  medium  must  be  absolute  so  that  no  space  is  left  for  air, 
otherwise  the  organisms  may  not  grow.  Roux  employs  a  long 
sterile  glass  tube,  which  he  completely  fills  with  melted  agar 


132         BACTERIOLOGICAL  LABORATORY  TECHNIC 

inoculated  with  the  organism  he  wishes  to  grow.  The  ends  of 
the  tube  are  then  sealed  in  a  bunsen  flame  and  there  being  no  air, 
anaerobic  conditions  are  fulfilled,  and  organisms  grow.  After 
colonies  appear  the  tube  is  broken  at  a  file-mark  near  the  colony 
and  tubes  inoculated  therefrom. 

Under  other  methods  large  Novy  jars  are  used  for  the  reception 
of  petri  dishes  and  test-tubes.  From  these  jars  the  air  is  with- 
drawn, and  hydrogen  allowed  to  flow  into  it.  A  solution  of 
pyrogallic  and  sodium  hydrate  is  placed  in  the  bottom  of  the  jar 
to  absorb  any  remaining  oxygen.  There  are  many  other  ingenious 
mechanical  ways  of  growing  bacteria  under  anaerobic  conditions 
and  the  student  is  referred  to  works  devoted  entirely  to  technique. 

Animal  Experiments 

To  determine  the  pathogenicity  of  bacteria;  to  measure  the 
strength  of  toxins  and  anti-toxins,  to  standardize  anti-toxins,  and 
to  recover  bacteria  in  pure  culture,  it  is  often  imperative  that 
small  laboratory  animals  be  used.  Guinea  pigs,  rabbits,  and 
mice  are  oftenest  employed.  Strong  young  animals  are  the  best. 
Culture  toxins  and  pathological  material  are  introduced  into  their 
bodies  in  various  ways.  A  favorite  one  is  to' shave  the  abdomen, 
scour  it  with  soap  and  water,  and  then  bichloride  of  mercury,  and 
finally  sterile  water.  With  a  pair  of  sterile  scissors  a  small  hole 
is  cut  in  the  abdominal  parieties  and  through  it  a  loop  containing 
a  drop  of  culture  is  run  into  the  peritoneal  cavity,  or  under  the 
skin.  With  ordinary  fluid  material,  a  syringe  may  be  used  to 
inject  it  directly  through  the  abdominal  wall.  The  animal  is 
carefully  weighed,  and  it  is  watched  from  day  to  day.  If  it  dies 
an  autospy  is  made  on  it. 

Other  methods  consist  in  injecting  fluid  culture  into  the  veins 
of  the  ear,  or  into  the  peritoneum,  by  means  of  sterile  hypoder- 
mic syringe.  The  autopsy  should  be  made  carefully,  the  animal 
should  be  thoroughly  wet  with  a  solution  of  bichloride  of  mercury, 


HISTOLOGICAL   METHODS  133 

then  it  should  be  stretched  over  a  pan,  especially  devised  for  the 
purpose,  or  nailed  to  a  board.  The  skin  over  the  abdomen  and 
thorax  must  then  be  shaved  and  sterilized  with  a  solution  of 
bichloride  of  mercury.  The  walls  should  then  be  seared  in  a  line 
from  the  throat  to  the  pubes  with  a  hot  knife,  and  through  this 
line  a  cut  should  be  made  opening  up  the  thoracic  and  abdominal 
cavities. 

By  means  of  a  hot  knife  spots  must  be  seared  on  the  various 
organs,  and  with  another  sterile  knife  cuts  should  be  made  into  the 
organs,  then  through  these  cuts  sterile  platinum  needles  are 
thrust,  and  then  culture  media  are  inoculated  with  them.  Some- 
times it  is  necessary  to  remove  bits  of  tissue  from  various  organs 
and  place  them  in  culture  media.  In  the  recovery  of  the  tubercle 
bacillus  from  animals  this  procedure  is  necessary.  Great  care 
must  be  taken  in  making  the  culture  and  all  tubes  should  be 
carefully  stored.  It  is  of  great  importance  to  make  smears  on 
cover-slips  as  well  as  cultures,  from  the  heart  cavities,  liver, 
kidneys,  peritoneal  cavity,  etc.,  and  stain  them  directly  with 
Gram  stain.  It  is  sometimes  necessary  to  inject  cultures,  or 
bits  of  nerve  tissue  from  a  rabies  case  into  the  brain.  To  do 
this,  remove  under  strict  aseptic  precautions,  a  button  of  bone 
from  the  skull  by  means  of  a  trephine.  It  should  not  be  forgotten 
that  animals  inoculated  and  killed  or  dying  after  infection  may 
present  dangerous  material  to  the  laboratory  personnel.  After 
an  autopsy,  a  strong  disinfectant  should  be  generously  spread 
over  all  parts  of  the  animal  and  autopsy  tray. 

Histological  Methods 

Sections  of  tissues  from  infected  animals  are  often  examined  and 
stained  by  appropriate  methods.  To  demonstrate  bacteria,  the 
tissues  should  be  hardened  in  alcohol  or  formaldehyde  solution 
(4  percent),  and  imbedded  in  celloidin,  then  cut  into  sections  and 
mounted  in  the  following  different  ways : 


134  BACTERIOLOGICAL  LABORATORY   TECHNIC 

I.  Loffler's  Method. 

(a)  Float  section  in  water. 

(6)  Remove  with  section  lifter  to  Loffler's  methylene  blue  from  five  to 
thirty  minutes. 

(c)  Decolorize  in  i  percent  solution  of  acetic  acid  for  ten  seconds. 

(d)  Dehydrate  in  absolute  alcohol  for  a  few  minutes. 

(e)  Clear  in  xylol. 

(/)   Mount  in  balsam. 

II.  Weigert's  Method. 

(a)  Place  in  lithium  carmine  five  minutes. 
(6)  Then  in  acid  alcohol  fifteen  seconds. 

(c)  Wash  in  water. 

(d)  Transfer  to  slide  and  dry  with  blotting  paper. 

(e)  Apply  Ehrlich's  gentian  violet  for  three  minutes. 
(/)   Blot  and  place  in  Gram's  solution  for  two  minutes, 
(g)  Wash  and  dehydrate  in  aniline  oil, 

(h}  Wash  with  xylol. 

(i)   Dry,  mount  in  balsam  and  examine. 

In  Loffler's  method  all  the  tissues,  especially  the  nuclei  and  the 
bacteria,  appear  blue. 

In  Weigert's  method,  if  the  bacteria  stain  by  Gram's  method, 
the  tissues  appear  pink,  the  bacteria  a  deep  blue-black.  Tubercle 
bacilli  are  to  be  stained  in  tissues,  best  fixed  in  formaldehyde  solu- 
tion, by  heating  with  carbol-fuchsin  as  given  on  page  106;  the  sec- 
tion is  decolorized  by  3-5  percent  hydrochloric  acid  and  cleared 
by  passing  it  through  95  percent  alcohol,  absolute  alcohol  and 
finally  xylol.  It  is  then  mounted  in  balsam. 

Parafnne  embedding  methods  may  be  employed,  but  for  these 
and  -other  methods  of  staining  the  student  is  referred  to  works 
solely  devoted  to  technique.  The  staining  methods  are  the 
same  for  paraffine  and  in  experienced  hands  give  better  results. 


CHAPTER  VII 
ANTISEPTICS  AND  DISINFECTANTS 

Many  chemical  substances  have  the  power  of  entering  into 
chemical  union  with  the  protoplasm  of  bacterial  cells  and  so 
forming  new  compounds,  and  coagulating  the  protoplasm; 
other  chemicals  dissolve  the  bacterial  bodies. 

Bacteria  differ  in  their  powers  to  resist  these  agencies;  the 
anthrax  spore  is  much  more  difficult  to  kill  than  the  typhoid 
bacillus;  these  chemical  substances  act  better  at  a  high  than  a  low 
temperature. 

A  chemical  disinfectant,  such  as  copper  sulphate,  acts  more 
rapidly  and  effectively  in  a  watery  solution  than  in  a  complex 
albuminous  one. 

Park's  division  of  the  change  of  viruses  under  the  influence  of 
chemicals  is  convenient  and  instructive.  Attenuation  is  the  tem- 
porary restriction  of  growth,  but  especially  of  virulence  and 
pathogenicity;  these  are  resumed  upon  cessation  of  action  of  the 
chemical.  Antisepsis  is  a  definite  restriction  of  growth  but  there 
is  no  destruction.  Incomplete  sterilization  is  the  destruction  of 
vegetative  forms  of  bacteria  but  not  spores.  Disinfection  or 
sterilization  is  the  destruction  of  all  disease  producing  virus. 

It  is  often  necessary  to  determine  the  exact  minimum  amount  of 
an  antiseptic  that  will  destroy  a  given  organism  or  produce  a 
complete  inhibition  of  growth;  for  this  purpose  small  amounts  of 
a  disinfectant  are  added  to  gelatine  in  test-tubes  and  these  are 
poured  into  plates  and  the  result  noted. 

Previous  to  pouring  the  plates  each  tube  is  inoculated  with  a 
loopful  of  culture  and  thoroughly  mixed  with  the  medium. 


136  ANTISEPTICS   AND   DISINFECTANTS 

Another  method  is  to  make  bouillon  cultures  of  an  organism  and 
add  to  each  a  certain  percentage  of  the  solution  of  the  antiseptic, 
and  abstract  every  few  minutes  after  the  addition  of  the  chemical 
one  loopful  of  the  mixture  and  inoculate  fresh  media. 

Pieces  of  thread  sterilized,  and  then  put  in  fluid  cultures  may 
be  used  in  experiments;  they  are  dipped  into  solutions  of  chemicals 
for  varying  lengths  of  time  and  then  placed  in  culture  media 
and  growth  noted. 

It  will  be  found  in  the  case  of  most  antiseptics  in  dilute  solution 
that  an  interval  of  time  must  elapse  before  the  organisms  are 
killed.  This  is  determined  by  observing  the  cultures  made  from 
the  mixture.  After  five  minutes,  growth  may  occur,  but  after 
one  hour,  all  may  be  dead,  or  it  may  take  two  or  three  hours. 

The  student  should  refer  to  works  on  hygiene  for  standard 
methods  of  controlling  disinfectants,  for  example  the  Hygienic 
Laboratory  and  the  Rideal- Walker  methods. 

The  most  valuable  chemical  disinfectants  are  those  that  kill  in 
highly  dilute  solution  in  a  short  time. 

Bichloride  of  mercury  is  a  highly  efficient  germicide  in  watery 
solutions;  if,  however,  albuminous  matter  is  present  its  action  is 
inhibited  very  much. 

CHEMICAL  DISINFECTANTS 

Mercury  Salts. — Bichloride  of  mercury  in  highly  dilute  solution 
is  a  very  valuable  antiseptic.  It  dissolves  in  16  parts  of  tepid 
water.  It  requires  an  acid  reaction  for  most  favorable  action 
and  the  tablets  now  on  the  market  are  made  up  with  some  acid 
having  no  effect  upon  the  mercury  salt.  In  i-ioo  water  solution 
this  salt  will  kill  anthrax  spores  in  twenty  minutes.  In  blood, 
the  anthrax  bacillus  is  killed  by  a  1-2,000  solution  in  a  few  minutes. 
In  bouillon  the  same  organism  is  killed  in  a  dilution  of  1-40,000; 
in  water,  1-500,000;  all  in  the  same  interval  of  time.  The  pres- 
ence of  the  albumins  in  the  blood  or  bouillon,  no  doubt  acts  as  a 


CHEMICAL   DISINFECTANTS  137 

protecting  envelope  about  the  bodies  of  the  bacteria,  being  there- 
fore unreliable  for  disinfecting  sputum  and  pus.  It  is  also  more 
useful  and  powerful  when  it  is  acidulated  with  a  5.  percent  of 
HC1,  or  when  it  is  mixed  with  common  salt  or  ammonium  chloride. 
In  culture  1^1,000,000  solution  prevents  the  growth  of  most 
pathogenic  bacteria.  Biniodide  of  mercury  is  said  by  some  ob- 
servers to  be  more  powerful  than  the  bichloride.  It  is  certainly 
less  likely  to  be  interfered  with  by  albumins. 

Sulphate  of  copper  in  water  is  a  powerful  germicide.  It  is  more 
potent  in  watery  solution  than  in  bouillon.  It  has  a  remarkable 
affinity  for  algae  and  for  moulds.  The  author  found  that  if 
moulds  are  put  into  alkaline  solution  of  copper  sulphate  and 
heated,  the  copper  enters  into  chemical  union  with  the  protoplasm 
of  the  mycelia,  hyphae,  and  the  spores;  1-400,000  of  copper  sul- 
phate in  water  destroys  the  typhoid  bacilli.  Even  nascent  copper 
kills  the  typhoid  bacilli,  so  that  copper  foil  in  drinking  water  has 
the  power,  after  a  few  hours'  contact,  of  destroying  bacteria  in 
the  water. 

The  silver  salts  are  useful  in  medicine  as  disinfectants,  especially 
on  mucous  surfaces.  The  nitrate  of  silver  is  one  of  the  most  valu- 
able of  all  preparations;  it  is  about  a  fourth  as  efficient  as 
bichloride  of  mercury  and  is  not  nearly  so  toxic.  Some  of  the 
albuminates  of  silver  are  useful  because  of  their  non-irritating 
action. 

Acids,  especially  the  mineral  ones,  are  valuable  disinfectants  in 
not  too  dilute  solutions.  They  act  chiefly  as  inhibitors  of  growth 
rather  than  destroyers  of  bacterial  cells.  In  the  healthy  stomach, 
hydrochloric  acid  acts  as  a  normal  disinfectant,  and  in  disease, 
where  it  is  absent,  it  must  be  added  in  order  to  prevent  decom- 
position of  food.  Boric  acid  is  useful  in  medicine  on  mucous 
membranes. 

The  halogens,  iodine,  bromine  and  chlorine,  are  active  agents 
for  the  destruction  of  bacteria.  The  cheapest  of  these  is  chlorine. 
It  acts  best  in  contact  with  moisture,  since  it  decomposes  the 


ANTISEPTICS   AND   DISINFECTANTS 

molecule  of  water  combining  with  the  hydrogen  to  form  free  HC1 
and  setting  free  oxygen. 

Dry  chlorine  gas  (45  percent)  failed  to  kill  dry  anthrax  spores 
in  one  hour,  but  when  moisture  was  introduced  4  percent  chlorine 
killed  the  spores. 

"  Chloride  of  lime ,"  chlorinated  lime,  in  i  percent  solution  kills 
most  bacteria  in  one  to  five  minutes.  Iodine  preparations  like 
chlorine  ones  are  very  powerful.  They  are  of  great  use  in  medi- 
cine; ordinary  tincture  of  iodine  painted  over  infected  areas  acts 
as  a  powerful  germicidal  agent.  It  is  too  expensive  to  use  in 
house  disinfection  and  it  is  exceedingly  destructive  to  all  metallic 
objects.  A  5  percent  solution  in  50  percent  alcohol  acts  as  a 
splendid  disinfectant  for  intrauterine  injection  in  puerperal  sepsis. 
It  is  now  said  that  10  percent  iodine  tincture  in  70  percent  alcohol 
is  the  most  efficacious,  practical,  medical  disinfectant.  Many 
claim  it  to  have  the  highest  penetrating  powers. 

Dakin's  solution  is  a  mixture  of  chlorinated  lime  and  sodium 
carbonate  in  water  and  sodium  bicarbonate,  with  an  alkalinity  of 
.45  percent.  This  mixture  when  in  the  presence  of  organic 
matter  decomposes  with  the  formation  of  hypochlorous  acid 
which  may  further  change  into  chlorimido — (NCI)  to  which 
changes  the  antiseptic  action  is  due.  The  solution  is  used  for 
infected  wounds,  being  introduced  by  methods  of  infiltration  and 
drainage.  It  has  an  irritant  effect  upon  the  skin  which  tissue 
must  be  protected.  The  germ-killing  power  of  this  solution  is 
very  high.  Its  use  was  followed  in  the  great  European  war,  by  a 
marked  reduction  in  spreading  and  fatal  infections  from  lacerated 
wounds. 

Chloramin  is  another  product  of  chlorinated  lime,  depending 
on  chlorin  for  its  action.  Being  less  toxic  it  may  be  applied  to 
tissues  or  even  mucous  membranes  in  2  percent  solution  but  it  is 
irritating  and  surrounding  surfaces  must  be  protected. 

DicUoramin  T.  still  a  chlorine  disinfectant,  is  less  irritating  than 
the  foregoing  but  being  unstable,  must  be  suspended  in  an  oil  like 


CHEMICAL   DISINFECTANTS  139 

eucalyptoe,  saturated  with  chlorine  gas.  It  can  be  sprayed  on  a 
surface  or  pressed  into  a  wound. 

Carbolic  acid  is  valuable  as  a  disinfectant  because  of  its  stability. 
A  1-1,000  solution  inhibits  bacterial  growth;  a  5  percent  solution 
is  a  thoroughly  reliable  disinfectant  for  morbid  discharges;  this 
strength  is  not  injurious  to  metals  or  fabrics.  A  thorough  solu- 
tion should  be  made,  and  to  be  very  efficient,  5  percent  HC1 
should  be  added  to  it. 

Cresol,  lysol  and  creolin  are  useful  as  disinfectants,  but  are 
sometimes  unreliable  since  perfect  solution  cannot  always  be 
made.  The  mixture  of  one  of  these  substances  with  water  is 
more  of  an  emulsion  than  solution.  Anthrax  spores  have  been 
known  to  live  for  hours  in  creolin  solutions.  The  value  of  these 
cresols  is  that  when  applied  to  a  surface  the  water  may  evaporate 
but  the  germicide  sticks  and  continues  its  effects.  Glycerine  is 
sometimes  added  to  lighter  phenol  solutions  to  assist  this  action. 

Peroxide  of  Hydrogen  has  a  great  reputation  in  medicine  as  an 
antiseptic.  It  kills  bacteria,  especially  the  pus  cocci,  in  a  few 
minutes  in  a  15  percent  solution.  A  40  percent  solution  will  kill 
anthrax  spores  in  a  few  hours.  It  is  a  powerful  agent  when  fresh, 
and  is  not  poisonous.  It  combines  with  organic  matter  and 
becomes  inert.  It  degenerates  if  exposed  to  atmosphere  and  if  it 
comes  in  contact  with  the  ferments  of  the  blood  (haemase). 

Formaldehyde  gas,  CH^O,  is,  by  all  means,  the  most  useful,  as 
well  as  the  most  powerful  disinfecting  agent  that  we  have.  In 
solution  40  percent  in  water,  it  is  known  as  formaline.  It  has  a 
marked  affinity  for  organic  substances  and  forms  chemical  com- 
binations with  many  organic  bodies.  When  it  unites  with  am- 
monia it  becomes  inert  until  some  acid  frees  it.  It  unites  with 
iron,  but  other  metals  are  unaffected.  Its  use  in  medicine  is  wide 
and  varied.  It  is  a  deodorizer;  renders  gelatine  glass-like  and 
insoluble  in  boiling  water.  It  may  be  liberated  as  a  gas  in  apart- 
ments and  ships,  actively  destroying  all  bacteria.  One  percent 
of  the  vapor  in  the  air  of  a  closed  room,  if  the  air  is  moist,  destroys 


140   „  ANTISEPTICS   AND   DISINFECTANTS 

bacteria  after  twelve  hours.  It  is  best  to  keep  the  room  closed 
for  twenty-four  hours.  It  may  be  thrown  into  the  room  in  many 
ways;  by  generators  which  decompose  the  vapor  of  wood  alcohol, 
when  they  reach  hot  platinum  sponges,  salt,  or  hot  copper;  by 
vaporizing  a  solution  by  means  of  heat;  by  adding  permanganate 
of  potash  to  a  solution  of  formaline;  by  spraying  a  concentrated 
solution  over  bedding,  floors,  and  walls,  then  closing  the  apart- 
ment. It  is  very  much  more  active  in  warm  air  than  in  cold,  and 
when  the  air  is  moist.  It  has  been  known  to  destroy  anthrax 
spores  wrapped  up  in  paper  and  placed  under  blankets.  All  of 
the  pathogenic  bacteria  are  killed  by  it,  the  Staphylococcus 
aureus  and  anthrax  spores  being  more  resistant  than  anything 
else.  It  will  not  kill  moulds  unless  highly  concentrated.  As  di- 
lute watery  and  alcoholic  solutions  decompose  they  should  only 
be  used  when  freshly  made. 

Sulphur  Dioxide  Gas. — An  old  and  rather  unreliable  form  of 
disinfectant.  It  does  not  kill  anthrax  spores  very  readily,  as 
it  requires  an  exposure  of  twenty-four  hours  to  a  40  percent  vapor 
in  a  room.  It  is  generated  by  burning  sulphur  in  a  room  tightly 
closed,  and  it  is  much  more  efficient  if  weter  is  vaporized  in 
the  room.  It  is  not  very  penetrating,  is  poisonous  to  breathe, 
speedily  bleaches  fabrics,  and  attacks  metal  objects.  It  is  much 
superior  to  formaline  as  an  agent  for  the  destruction  of  insects, 
especially  mosquitoes,  also  to  kill  rats  infected  with  plague  bacilli. 

Lime. — Ordinary  quick  lime,  or  whitewash,  is  highly  germicidal. 
It  is  especially  efficacious  in  disinfecting  feces  from  typhoid  cases. 
Typhoid  bacilli  are  killed  after  one  hour's  exposure  to  a  20  percent 
mixture. 

Potassium  permanganate  in  3  percent  solution  is  said  by  Koch 
to  kill  anthrax  spores  in  twenty-four  hours.  It  is  not  so  efficient 
a  germicidal  agent  as  supposed. 

Turpentine  and  essential  oils  are  efficient  germicides  in  con- 
centration. Common  mustard  rubbed  in  the  hands  is  said  to 
make  them  sterile. 


CHEMICAL   DISINFECTANTS  141 

Alcohol. — Ninety-five  percent  and  absolute  alcohols  are  not 
antiseptic  for  the  anthrax  spores,  since  they  will  live  for  many 
hours  in  contact  with  absolute  alcohol.  In  general  it  is  unreliable. 
Seventy  percent  alcohol  is  the  most  efficient  strength. 

Zinc  chloride  in  concentration  is  a  powerful  germicide.  A  2  per- 
cent solution  will  kill  the  ordinary  pyogenic  bacteria  in  two  hours. 

Sputum,  urine  and  dejecta  are  best  disinfected  by  heat.  Chem- 
icals often  are  inert  because  they  cannot  penetrate  the  albumi- 
nous masses  of  the  sputum  or  feces.  Long  contact  with  carbolic 
acid  acidulated  with  HC1  is  very  efficient.  Concentrated  forma- 
line and  solutions  of  chloride  of  lime  may  be  used,  also  a  heavy 
mush  of  lime  in  water. 

Boiling  or  heating  instruments  and  dressings  by  high  moist  heat, 
as  in  an  autoclave,  is  the  most  reliable  method  of  rendering  them 
sterile.  The  exposure  of  dressings  to  i5o°C.  for  one  hour,  or 
boiling  instruments,  thoroughly  cleaned  mechanically,  for  twenty 
to  thirty  minutes  makes  them  certainly  sterile. 

Disinfection  of  the  skin  is  a  difficult  undertaking  from  a 
bacteriological  standpoint.  In  the  deep  layers  of  the  skin,  and  in 
the  sweat  glands  and  hair  follicles,  bacteria  often  exist,  even  after 
the  most  thorough  and  prolonged  disinfection.  The  application 
of  soap  and  water  with  a  stiff  brush  is  by  all  means  the  most 
valuable  part  of  the  process,  since  with  the  removal  of  the  dirt 
most  of  the  bacteria  are  removed.  Thorough  scrubbing  with  soap 
and  sterile  water,  followed  by  scrubbing  with  a  1-1,000  bichloride 
solution,  cleansing  the  nails  with  a  sterile  brush,  and  prolonged 
immersion  in  bichloride  or  permanganate  of  potash  solution, 
complete  the  process.  Modern  methods,  even  after  all  this 
preparation,  require  the  use  of  rubber  gloves  that  have  been 
sterilized  by  boiling.  The  faultiest  part  of  the  preparation  for  an 
aseptic  operation  from  a  bacteriological  standpoint,  has  always 
been  considered  to  be  the  sterilization  of  the  hands,  and  if  these 
can  be  covered  by  rubber  gloves  that  are  sterile,  the  fault  can  be 
surely  eliminated. 


142 


ANTISEPTICS   AND   DISINFECTANTS 


Antiseptic  Values  (after  Park). 

The  figures  refer  to  the  relative  antiseptic  powers  of  various 
agents  for  fluids  containing  organic  matter. 

Alum i  to      222 

Aluminium  acetate i  to    6,000 

Ammonium  chloride to          9 

Boric  acid to       143 

Calcium  chloride : to         25 

Calcium  hypochlorite to    1,000 

Carbolic  acid to       333 

Chloral  hydrate to       107 

Copper  sulphate to    2,000 

Ferrous  sulphate to       200 

Formaldehyde,  40  percent to  10,000 

Hydrogen  peroxide to  20,000 

Mercuric  iodide to  25,000 

Mercuric  chloride to  40,000 

Quinine  sulphate to      800 

Silver  nitrate to  12,500 

Zinc  chloride to       500 

Zinc  sulphate to        20 


CHAPTER  VII 

BACTERIA 
STREPTOCOCCUS  PYOGENES 

Streptococcus  Pyogenes. 

Streptococcus  Erysipelatis.    Chain  Coccus  (Fig.  36). 
Morphology  and  Stains. — Cocci  grow  in  catenate  form  of  from 
4  to  40  individuals  to  a  chain.    The  cocci  divide  in  a  single  plane, 


FIG.  36.— Streptococcus  pyogenes.     (Kolle  and  Wassermann.) 


by  transverse  fission  thus  giving  rise  to  chains.  The  cocci  are 
not  motile,  and  do  not  have  spores.  They  can  be  stained  with 
all  basic  stains,  and  retain  the  Gram's  stain. 

Relation  to  Oxygen. — They  grow  either  in  the  presence  or 
absence  of  oxygen,  and  are,  therefore,  facultative  aerobes. 

Temperature  and  Food  Requirements. 

Develop  best  at  37°C.  Will  not  grow  at  47°C.  Never  vege- 
tate luxuriantly  on  any  culture  media,  but  are  most  prolific  on 

143 


144  BACTERIA 

one  that  is  faintly  acid  and  contains  animal  juices  like  serum. 
They  must  be  transplanted  frequently.  On  gelatine  they  grow 
scantily  without  liquefaction,  the  growth  consists  of  discrete  little 
masses,  while  on  agar  they  appear  translucent  colonies  of  very 
small  grayish  granula.  In  bouillon  cultures  some  varieties  either 
cloud  the  medium  uniformly,  or  else  sedimentate  in  the  form  of 
little  balls,  the  supernatant  fluid  remaining  clear.  It  ferments 
some  simple  sugars  but  does  not  form  gas.  In  milk  the  growth  is 
more  luxuriant,  and  becoming  acid,  may  be  coagulated  in  twenty- 
four  hours.  On  potato  the  growth  is  invisible  and  scanty,  or 
absent.  On  blood  agar  plates  colonies  appear  as  tiny  gray 
points  with  a  zone  of  hemolysis  about  them.  Sugar  media 
of  the  simpler  carbohydrates,  show  acidification. 

Vital  Resistance. — Thermal  death-point  is  54°C.  in  five  min- 
utes. Virulence  in  dried  albuminous  matter  (pus)  is  retained  for 
months.  If  kept  on  ice,  vitality  and  virulence  are  retained  for 
months  also. 

Chemical  Activities. — Lactic  acid  and  sulphuretted  hydrogen  are 
produced,  also  ferments  which  have  the  property  of  dissolving 
fibrin  under  anaerobic  conditions.  They  are  also  capable  of  dis- 
solving red  blood  corpuscles,  either  in  culture  media  or  in  the 
body  and  about  cultures  on  blood  agar  plates  there  is  a  clear  halo 
of  hemolysis,  streptocolysin.  They  produce  a  strong  soluble 
toxin,  which  can  be  filtered  from  the  bouillon  and  precipitated 
with  alcohol.  This  causes  necrosis,  anaemia  and  death. 

Habitat. — In  sewage,  dwellings,  dust,  on  the  healthy  human 
body,  and  in  the  cavities  of  the  respiratory  tract,  vagina,  rectum, 
and  in  the  faeces.  It  is  the  cause  of  many  diseases,  i.e.,  erysipelas, 
puerperal  fever,  meningitis,  pneumonia,  endocarditis,  peritonitis, 
tonsillitis,  osteomyelitis,  and  the  diarrhoea  of  children. 

In  general  septicaemia  streptococcus  is  found  in  the  blood,  and 
plays  an  important  role  in  secondary  infection,  causing  an  aggra- 
vation of  the  original  infection,  and  often  death.  It  is  especially 
active  in  phthisis,  scarlatina,  small-pox,  and  diphtheria,  in  which 


STREPTOCOCCUS   PYOGENES  145 

diseases  it  is  often  the  cause  of  death.  Many  of  the  symptoms 
of  phthisis  are  due  to  the  toxins  of  the  streptococcus;  cavity  for- 
mation and  hectic  fever  for  example.  Its  virulence  can  be  in- 
tensified by  passing  it  through  a  series  of  animals,  until,  finally, 
M>ooo  cu.  mm.  kills  in  one  day  all  the  mice  injected  with  this 
dose.  The  toxin  contains  a  peculiar  haemolytic  substance,  which, 
as  before  remarked,  dissolves  red  cells  of  the  blood,  hence  the 
anaemia  in  septicaemia  and  in  suppuration.  The  toxin  of  the 
streptococcus,  if  injected  under  the  skin,  causes  redness  like  ery- 
sipelas. Coley's  fluid  containing  this  toxin  is  used  to  treat 
sarcomata,  since  infection  with  the  streptococcus  has  been  known 
to  cause  a  disappearance  of  these  tumors.  Practically  all  animals 
are  susceptible  to  the  streptococcus. 

Agglutinations. — The  serum  from  an  animal  injected  with  strep- 
tococci, or  immunized  against  it,  will  agglutinate  streptococci. 

Anti-toxic  sera  have  been  prepared  by  injecting  horses  with 
highly  virulent  living  culture  of  streptococci.  The  serum  protects 
to  a  limited  degree,  and  has  some  curative  properties.  Cultures 
of  cocci  from  human  sources  have  been  found  to  produce  the 
best  toxins;  there  are,  however,  many  strains. 

The  foregoing  description  represents  the  principal  characters  of 
the  most  important  member  of  a  large  group  of  closely  related 
streptococci.  Distinctions  in  the  group  are  based  upon  the 
solution  of  red  blood  cells,  the  fermentation  of  lactose,  of  maltose, 
of  salicin  and  the  coagulation  of  milk.  The  relative  value  of 
each  of  the  members  is  not  yet  settled  but  investigations  have 
enabled  laboratory  workers  to  elaborate  certain  techniques 
which  may  be  expected  to  clear  up  the  matter.  The  varieties 
now  recognized  are  Str.  hemolyticus,  epidemicus,  anginosus, 
fecalis,  salivarius,  equinus,  and  mitis.  Some  of  these  organisms 
produce  no  hemolysis;  they  come  under  the  term  non-hemolyticus 
and  are  of  importance  in  certain  respiratory  and  allied  diseases. 
There  is  a  group  of  organisms,  a  sort  of  connecting  link  with 
pneumococci,  which  produce  green  colonies  on  blood  agar  and 
10 


146  BACTERIA 

are  called  Str.  viridans.  These  are  to  be  distinguished  from 
true  pneumococci  by  the  formation  of  a  tiny  rim  of  blood 
clearing  in  blood  plates  and  by  their  insolubility  in  bile.  These 
and  the  group  of  non-hemolytic  streptococci  are  held  responsible 
for  some  cases  of  arthritis,  endocarditis,  sinusitis,  nephritis,  etc 

PNEUMOCOCCUS 

Streptococcus  pneumonias  commonly  known  as  the  pneumo- 
coccus,  or  Diplococcus  lanceolatus  (Fig.  39).  (For  types  see 
page  76.) 

Morphology  and  Stains. — This  organism  is  usually  found  in  the 
tissues  and  sputum,  in  the  form  of  lance-shaped  cocci,  surrounded 
by  a  capsule.  Is  almost  always  associated  in  pairs,  though  some- 
times in  chains  of  five  or  six  members.  In  albuminous  fluids,  or 
blood  serum,  and  in  milk,  the  organism  exhibits  a  well-defined 
capsule;  in  bouillon  and  other  media,  it  loses  the  capsule  and  the 
lanceolate  shape,  and  often  appears  spherical,  in  pairs,  or  chains. 
It  is  not  motile,  has  no  flagella  or  spores,  is  easily  stained  by  all 
the  basic  aniline  dyes,  and  keeps  the  color  by  Gram's  method. 
Under  certain  conditions  it  strongly  resembles  the  streptococcus 
pyogenes,  and  may  be  differentiated  therefrom  by  growing  it  on 
agar  smeared  with  blood.  The  streptococcus  causes  a  haemolysis 
of  the  corpuscles,  while  the  pneumococcus  does  not  and  the 
colonies  are  greenish. 

Oxygen  Relations. — It  is  a  facultative  aerobe. 

Grows  rapidly,  but  never  luxuriantly  at  37.5°C.;  at  22°C.  much 
more -slowly,  often  not  at  all.  Grows  better  in  the  presence  of 
serum  or  haemoglobin. 

Vital  Resistance. — Easily  killed  at  a  temperature  of  52°C.,  ex- 
posed for  ten  minutes.  Direct  sunlight  also  kills  it  in  twelve 
hours.  While  it  quickly  dies  on  ordinary  culture  media,  it  may 
live  in  dried  sputum  or  pus  exposed  to  diffuse  light  and  desiccation, 
for  four  months. 


PNEUMOCOCCUS 


147 


Cultures. — On  gelatine  plate  it  produces  very  minute  colonies 
after  quite  a  length  of  time.  On  glycerine  agar  it  grows  better, 
but  the  colonies  are  small  and  difficult  to  see.  In  both,  the  colo- 
nies are  whitish,  with  a  pearly  lustre.  On  blood  serum  it  grows 
in  transparent  colonies.  On  blood  agar  the  colonies  are  tiny  and 


FIG.  37.  —  Diplococcus  pneumonias,  from  the  heart's  blood  of  a  rabbit.     X 
1,000.     (Frankel  and  Pfeiffer.) 


of  a  greenish  color,  lying  on  a  brown  base  due  to  production  of 
methemoglobin.  In  bouillon  it  grows  feebly,  with  a  whitish 
sediment,  and  in  the  form  of  chains.  Here  the  growth  is  inhibited 
by  the  products  of  its  own  metabolism,  i.e.,  lactic  acid.  If  this 
is  neutralized  by  putting  chalk  into  the  bouillon  the  growth 
becomes  luxuriant  and  the  bouillon  becomes  thick.  On  potato  it 


148  BACTERIA 

will  not  grow.  It  ferments  some  of  the  sugars,  the  most  impor- 
tant and  characteristic  being  inulin.  No  gas  is  formed.  Pneu- 
mococcic  are  soluble  in  bile  or  a  solution  of  its  salts,  a  distinguishing 
determinative  character. 

Habitat. — Outside  the  human  body  it  has  not  been  found,  but 
is  normally  present  in  the  mouth  of  about  30  percent  of  all  people. 
In  apparent  health  cultures  from  the  throat  or  the  sputum  in- 
into  animals  often  causes  pneumococcic  septicaemia  because 
Type  IV  (see  page  76)  may  be  frequently  found  in  them.  The 
so-called  "fixed  types"  I,  II,  and  III  occur  in  the  throat  during 
pneumonia  but  disappear  shortly  after  recovery.  It  also  may  be 
found  on  the  conjunctiva  and  nose  in  health. 

Chemical  Activities. — No  soluble  toxin  has  been  discovered. 
The  toxic  properties  are  due  to  an  endo-toxin.  This  organism  is 
a  pyogenic  one,  and  causes  dense  fibrinous  exu dates  on  serous 
membranes.  All  tissues  of  the  body  may  be  attacked.  Some 
strains  of  pneumococci  are  more  neurotoxic  than  others. 

In  rabbits  an  intravenous  injection  of  pneumococci  will  cause 
a  septicaemia  with  at  times  areas  of  lobular  pneumonia.  The 
only  successful  reproduction  of  pneumonia  in  the  lower  animals 
is  accomplished  by  tracheal  insufflation  of  pure  virulent  cultures. 
In  human  infection  the  organisms  are  forcibly  inhaled  into  the 
deepest  recesses  of  the  lungs.  Pneumonia  may  be  haematogenous 
in  origin  also. 

Besides  pneumonia,  any  serous  membrane  may  be  attacked  and 
pleuritis,  peritonitis,  pericarditis,  or  meningitis  may  be  caused. 
Abscesses  anywhere  may  be  due  to  the  pneumococcus.  Mucous 
membranes  of  the  throat  often  are  affected;  middle  ear  abscesses 
also  may  be  caused  by  this  organism.  Pneumococcic  septicaemias 
are  common. 

During  pneumonia,  pneumococci  may  be  recovered  from  the 
blood  before  the  crisis  by  means  of  blood  cultures;  10  c.c.  of  blood 
abstracted  from  veins  is  mixed  with  500  c.c.  of  milk  or  bouillon 
and  incubated.  In  twenty-four  hours  pneumococci,  if  present, 


COCCUS    OF   MENINGITIS  149 

grow  luxuriantly.  Just  before  the  crisis  the  organisms  will  not 
grow. 

Immunity  and  Susceptibility. — The  susceptibility  of  man  varies 
greatly.  Exposure  to  cold  and  hardships  of  various  kinds  predis- 
pose to  pneumonia.  One  attack  does  not  prevent  another.  It 
has  been  observed  that  normal  leucocytes  only  become  phagocytic 
toward  the  pneumococcus  when  lying  in  anti-pneumococci  serum. 
It  has  even  been  noticed  that  these  organisms  grow  better  in  the 
anti-serum,  rather  than  in  the  normal  serum.  Animals  have  been 
immunized  by  injecting  cultures  and  toxin.  The  immune  serum 
thus  produced  protects  small  animals  against  infection,  and  stimu- 
lates phagocytosis.  It  has  been  used  therapeutically  in  man  for 
the  cure  of  pneumonia  with  hopeful  results.  Oleate  of  soda  aids 
in  bacteriolysis  of  pneumococci  by  sera,  if  added  to  the  various 
varieties  of  immune  sera  (see  page  76).  Most  mammals,  but  few 
if  any  birds  are  susceptible  to  the  pneumococci;  mice,  being 
very  easily  infected,  are  used  for  isolation  purposes. 

Agglutination  of  pneumococci  is  caused  by  the  blood  of  infected 
individuals,  even  diluted  at  1-60.  Immune  serum  also  has  the 
same  action. 

Opsonins  increase  during  the  course  of  pneumonia  and  are  at 
their  height  at  or  just  after  crisis. 

Pneumococcus  mucosus,  also  called  Type  III  is  distinguished 
by  its  large  size,  long  chains,  capsule,  more  generous  growth 
in  large  moist  colonies  of  not  such  a  distinct  green  color  and  its 
ability  to  produce  a  serious  form  of  pneumonia. 

COCCUS  OF  MENINGITIS 

Micrococcus  Meningitidis. 

Diplococcus  intracellularis  meningitidis. 

Meningococcus  (Fig.  38). 

This  organism  is  the  cause  of  epidemic  cerebro-spinal  meningitis. 

Morphology  and  Stains. — Resembles  the  gonococcus  closely, 


150 


BACTERIA 


because  it  grows  in  biscuit-shaped  pairs;  is  nearly  always  within 
pus  cells,  and  like  the  gonococcus  it  is  decolorized  by  Gram's 
stain.  It  has  no  spores  or  flagella;  is  not  motile;  grows  in  short 
chains  at  times,  and  on  ordinary  media  best  at  37°C. 


Q 


V 


FIG.  38. — Meningococcus  in  spinal  fluid.     (From  Hiss  and  Zinsser's  Bacteri- 
ology, Copyright  by  D.  Appleton  &  Co.) 


Relation  to  Oxygen. — It  can  be  cultivated  from  the  meninges 
at  first  best  under  conditions  of  low  oxygen  tension,  as  in  a  Novy 
jar  or  in  an  atmosphere  of  carbon  dioxide  and  moisture;  once 
growing  in  the  laboratory  it  is  more  luxuriant  in  aerobic  tubes. 

Vital  Resistance. — It  is  killed  after  ten-minutes'  exposure  to 


COCCUS   OF   MENINGITIS  I$I 

65°C.  and  is  easily  destroyed  by  drying,  and  by  light.  It  dies 
out  rapidly  on  artificial  culture  media. 

Cultures. — Best  isolated  on  neutral  semi-solid  ascitic  fluid  or 
serum  agar  in  a  moist  chamber.  Growth  is  pale  gray  white 
translucent  moist  separate  colonies.  On  glycerine  agar  it  grows, 
sparingly  as  white  viscid  colonies;  occasionally  it  develops  on 
potato  ;  thrives  on  blood  serum,  especially  if  smeared  with  blood, 
and  does  not  liquefy  the  serom. 

Habitat. — It  is  found  in  the  pus  from  the  meninges,  sputum, 
and  nasal  mucus  of  persons  afflicted  with  epidemic  meningitis,  or 
spotted  fever.  It  has  been  found  in  the  mucous  membranes  of 
healthy  individuals,  and  these  persons  may  be  "carriers"  of  infec- 
tion. After  spinal  puncture,  it  may  be  seen  in  the  pus  cells,  and 
the  diagnosis  of  the  disease  can  be  made  in  this  way. 

Virulence. — It  is  scarcely  virulent  for  lower  animals.  If  given 
by  hypodermics  into  the  pleura,  or  peritoneum,  it  produces  death 
in  mice.  Meningitis  may  be,  in  monkeys,  produced  by  subdural 
injection. 

Chemical  Activities. — Produces  an  en»do-toxin  but  no  soluble 
toxin.  It  is  not  chromogenic. 

Agglutination  is  caused  by  immune  serum  and  it  is  upon  their 
property  that  anti-sera  are  standardized.  It  has  been  discovered, 
by  means  of  this  anti-body  and  by  the  failure  of  certain  sera  to  do 
good,  that  there  are  several  types  of  meningococci  of  different 
antigenic  qualities.  Anti-sera  are  made  now  with  all  available 
varieties. 

Method  of  Infection. — The  infection  atrium  of  the  coccus  is 
not  certainly  known  but  most  of  the  evidence  points  to  the  nasal 
passages  and  cribriform  plate  to  the  subdural  space. 

Specific  Therapy  is  practicable;  it  has  been  discussed  just  above 
and  on  page  76. 

There  is  another  important  Gram-negative  diplococcus  in  the 
nose  called  Micrococcus  catarrhalis.  It  is  differentiated  from 
the  meningitis  organism  by  its  free  growth  on  agar,  its  sugar  reac- 


152  BACTERIA 

tions  and  absence  of  active  pathogenic  properties.  It  will  appear 
on  nearly  all  plates  made  from  sputum  and  throat  cultures,  in 
crowded  city  life  of  temperate  zones.  It  seems  not  to  be  able  to 
incite  an  infection  but  to  continue  or  aggravate  one  already  under 
way. 

STAPHYLOCOCCUS  PYOGENES  AUREUS 

Staphylococcus  Pyogenes  Aureus  (Fig.  39). 

Micrococcus  Pyogenes. 

Staphylococcus  pyogenes  aureus,  albus,  and  'citreus  are  known 
commonly  as  Staphylococcus,  or  grape  coccus.  They  differ  only 
in  color  production  on  artificial  media. 


FIG.  39. — Staphylococcus  aureus.     (Williams.) 

The  Micrococcus  pyogenes  aureus  only  is  here  described;  the 
other  varieties  have  similar  but  much  feebler  pathogenic  powers. 

Morphology  and  Stains. — Round  cocci,  often  growing  in 
bunches  like  grapes.  Individual  cocci  dividing  in  two  planes. 
They  stain  very  well  with  all  basic  dyes,  and  are  not  decolorized 


STAPHYLOCOCCUS  PYOGENES  AUREUS 


153 


by  Gram's  method.     They  are  not  motile;  have  neither  flagella 
nor  spores. 

Oxygen  Requirements. — The  coccus  grows  well  in  oxygen,  and 
poorly  without  it. 

Temperature  and  Vital  Resistance. — 
Thrives  best  at  body  temperature,  but 
grows  well  at  room  temperature.  Resists 
drying  for  over  one  hundred  days  in  pus. 
Dry  thermal  death-point  is  8o°C.  for  one 
hour.  Moist  heat  7o°C.,  kills  in  ten  to 
twenty  minutes.  Resists  freezing  tempera- 
ture for  many  months. 

Exceedingly  resistant  to  formaldehyde, 
more  so  than  some  spore-bearing  organisms. 
Resists  light  also.  It  is  killed  by  corrosive 
sublimate  i-iooo  in  fifteen  minutes;  i  per- 
cent H2C>2  in  thirty  minutes. 

Chemical  Activities. — Produces  a  golden- 
yellow  pigment  only  under  oxygen.  Gener- 
ates acids,  but  no  free  gases.  Creates  indol 
and  sulphuretted  hydrogen;  ferments  urea, 
and  produces  ferments  that  dissolve  gelatine, 
and  the  coagulated  proteids  of  milk.  The 
toxin  is  soluble  in  water,  and  acts  intensely, 
causing  violent  local  reaction.  If  in  the  ab- 
dominal cavity,  it  causes  peritonitis.  Subcu- 
taneously  it  may  produce  sterile  abscess,  or 
local  necrosis.  There  is  produced  in  cultures 
a  toxin  having  a  destructive  action  upon  leucocytes  and  red 
blood  cells. 

Cultures. — In  gelatine  it  rapidly  forms  golden-yellow  colonies, 
that  quickly  liquefy  the  gelatine  (Fig.  40).  Sterile  products 
of  the  growth  also  liquefy  gelatine.  On  gelatine  plate,  yellowish 
to  orange  colonies  are  formed.  On  agar  streak  a  luxuriant  orange 


FIG.  40. — Gelatine 
culture  staphylococ- 
cus  aureus  one  week 
old.  (Williams.) 


154  BACTERIA 

growth  develops.  In  bouillon  there  is  a  marked  even  cloudiness, 
with  a  fine  pellicle  on  surface;  moderate  sediment,  which  upon 
shaking  is  broken  up.  Milk  is  rendered  acid  and  curdles  very 
soon,  the  curd  being  digested  finally. 

Potato  cultures  are  dry,  whitish  then  yellow,  and  finally  deep 
orange. 

Habitat. — Widely  distributed;  found  in  dirty  water,  sewage,  air, 
dust  of  streets  and  houses;  also  upon  the  skin;  normally  present  in 
the  mouth,  nose,  rectum,  anterior  urethra,  vagina,  and  external  ears. 

Pathogenesis. — In  man  it  is  the  cause  of  carbuncles,  abscesses, 
osteomyelitis,  septicaemia,  puerperal  infection,  and  any  inflamma- 
tion of  the  serous  membranes.  It  causes  acne  and  boils;  can,  and 
does  attack  any  tissue  of  the  body.  Endocarditis  is  a  very  grave 
affection  that  is  caused  by  this  organism.  It  also  plays  an 
important  role  in  secondary  infection,  causing  necrosis  of  pre- 
viously infected  tissues  (tubercles)  and  is  active  in  small-pox  and 
diphtheria.  Experimental  endocarditis  has  been  produced  in 
animals  by  injecting  it  into  the  veins.  By  passage  through 
animals  it  is  rendered  highly  virulent.  In  young,  diabetic  and 
anaemic  subjects,  its  action  is  often  rapidly  fatal.  Its  pathogenic 
action  is  often  wide  and  disastrous.  By  growing  it  under  an  ae- 
robic conditions  its  virulence  may  be  intensified,  and  the  ac- 
tivity with  which  it  liquefies  gelatine  is  an  index  of  its  malignancy. 

In  man  acne,  boils,  and  carbuncles  have  followed  the  rubbing  of 
culture  into  the  skin. 

Immunity. — Careful  injections  may  result  in  the  immunization 
of  the  lower  animals.  An  anti-serum  with  opsonic,  agglutinative, 
lytic  and  anti-toxic  properties  has  been  produced  and,  if  used 
fresh,  seems  to  have  a  slight  beneficial  effect  upon  staphylococcus 
septicemia.  Too  little  is  known  for  definite  statements.  Bac- 
terins  made  from  this  germ  have  been  used  with  excellent  results 
in  all  but  the  very  aggravated  and  fulminating  affections  caused 
by  it.  Bacterin  treatment  of  acne  and  furunculosis  has  estab- 
lished itself  as  most  efficacious. 


GONOCOCCUS 


155 


There  is  a  member  of  this  group  infesting  the  deep  layers  of  the 
skin  called  Micro,  epidermidis  albus.  It  is  of  feeble  pathogenic 
power,  but  may  delay  the  healing  of  surgical  wounds. 

GONOCOCCUS 

Micrococcus  Gonorrhoeas  (Neisser). 

Diplococcus  Gonorrhoea,  commonly  called  the  gonococcus  (Fig.  41). 


FIG.  41. — Gonococci  and  pus  cells.     X  1000.     (MacNeal.) 

Morphology  and  Stains. — The  morphology  of  this  organism  is 
peculiar  and  characteristic.  Always  found  in  pairs  which  are 
cemented  by  an  invisible  substance.  These  pairs  resemble  coffee 
beans  with  the  concave  sides  opposite  each  other  and  slightly  apart; 
or  kidneys  placed  with  the  hilums  facing  each  other. 

In  pus  it  is  generally  found  within  the  protoplasm  of  the  leuco- 
cytes, about,  though  never  within,  the  nuclei.  It  is  non-motile; 
has  no  flagella,  or  spores,  and  stains  readily  with  all  the  basic  stains; 
but  best  with  Loffler's  blue.  It  is  decolorized  by  Gram's  stain. 


156  BACTERIA 

This  point  is  most  important  in  differentiating  it  from  other 
diplococci,  except  the  meningococcus.  A  diplococcus  is  said  to 
exist  normally  in  some  urethras  that  resembles  the  gonococcus, 
but  is  Gram-positive. 

Oxygen  Requirements. — It  is  a  facultative  anaerobe. 

Vital  Conditions. — It  is  cultivated  with  difficulty  in  culture 
media.  Grows  best  at  about  37°C.  As  it  dies  quickly  in  usual 
culture  media,  a  special  one  must  be  employed;  that  containing  as- 
citic  or  hydrocele  fluid,  blood  or  urine  is  best.  It  does  not  with- 
stand high  temperature,  drying,  or  light,  very  long,  and  is  very 
easily  killed  in  culture  by  silver  salts.  In  tissues  of  the  urethra  it 
may  live  many  months. 

Cultures. — On  agar,  containing  ascites  fluid,  it  grows  very  spar- 
ingly. The  colonies  are  exceedingly  delicate,  and  gray,  turning 
to  yellowish,  and  are  scarcely  above  the  culture  media.  It  will  not 
grow  in  gelatine,  milk,  or  ordinary  bouillon,  but  in  one  made  of 
nutrose,  serum,  beef-extract,  and  peptone. 

Habitat. — Never  found  outside  the  human  organism,  except  on 
linen,  towels,  instruments,  etc.  It  is  in  all  senses  a  strict  parasite. 

Bacterial  Activities. — Apparently  does  not  produce  a  soluble 
toxin,  but  an  endo-toxin  (gonotoxin) ,  which  is  highly  resistant  to 
heat. 

Pathogenic  Virulence. — This  organism  does  not  infect  any  of 
the  lower  animals.  The  "gonotoxin,"  if  injected  into  small  ani- 
mals, produces  a  doughy  infiltrated  area,  which  undergoes  necrosis. 
It  has  been  found  that  filtrates  of  old  cultures  (sterile),  if  placed  on 
urethral  mucous  membranes,  can  produce  suppuration.  In  man, 
the  organism  causes  a  distressing  disease  (gonorrhoea),  which  may 
become  a  dangerous  one,  ending  even  in  death.  It  may  produce 
violent  inflammation  of  the  urethra  vagina,  uterus  fallopian  tubes, 
and  the  peritoneum.  It  frequently  affects  the  conjunctive,  and 
sometimes  causes  a  pan-ophthalmia,  which  destroys  the  sight.  It 
may  be  a  cause  of  plastic  arthritis  gonorrhceal  rheumatism,  endo- 
carditis, pleuritis.  In  fact,  any  serous  membranes  may  be  infected, 


MICROCOCCUS    TETRAGENUS  1 57 

and  very  serious  results  follow.  Cystitis  caused  by  the  gonococcus 
is  sometimes  followed  by  infection  of  the  kidneys.  In  the  urethra, 
the  cocci  may  burrow  deep  beneath  the  epithelial  cells,  and  set  up 
a  metaplasia,  or  abscess  formation.  The  purulent  exudate  is  rich 
in  phagocytes  gorged  with  cocci,  often  as  many  as  40  being  found 
within  a  cell. 

Immunity . — One  infection  does  not  confer  immunity  against 
further  infection.  There  is  no  reliable  means  of  producing  artifi- 
cial immunity.  However,  gonococcus  bacterins  are  of  some  value 
for  chronic  gonorrhoea.  Torrey  has  been  able  to  obtain  from 
rabbits  an  anti-serum  of  therapeutic  value  in  gonorrhoeal  arthritis. 

MICROCOCCUS  TETRAGENUS 

Micrococcus  Tetragenus. 

Morphology  and  Stains. — Round  or  oval  cocci;  found  in  pairs; 
more  commonly  in  fours  differing  in  size.  In  culture  this  form  of 
growth  is  apt  to  vary,  and  not  to  be  characteristic.  In  sections  of 
human  or  animal  tissues,  tetrads  only  are  found  that  are  always 
surrounded  by  a  capsule  which  is  stained  easily  by  eosin.  The 
cocci  are  stained  by  Gram's  method.  It  is  not  motile,  and  does 
not  form  spores. 

Oxygen  Requirements. — It  grows  very  well  in  the  presence  of 
oxygen,  and  poorly  without  it. 

Cultures. — Grows  well  on  all  common  culture  media.  On  gela- 
tine plates  its  growth  is  characterized  by  small  white  colonies, 
elevated,  with  sharp  outlines.  It  does  not  liquefy  the  gelatine. 
On  agar  it  grows  even  more  luxuriantly  than  on  galatine.  In 
bouillon  it  thrives  well,  depositing  a  heavy  precipitate.  In  milk 
it  causes  acidity  but  no  coagulation.  On  potato  it  also  grows, 
leaving  a  silvery  streak  where  the  inoculating  needle  was  drawn. 

Chemical  Activities. — It  produces  acid  in  sugar  bouillon,  but 
does  not  form  gas,  indol,  or  H2S. 

Habitat. — Has  never  been  found  outside  the  human  body;  is 


158  BACTERIA 

normally  present  in  the  saliva,  sputum  of  tuberculous  subjects,  in 
the  cavities  of  phthisical  lungs,  and  in  abscesses. 

Pathogenesis. — While  causing  a  fatal  septicaemia  in  mice,  and 
abscesses  in  rabbits,  it  is  not  of  much  moment  from  a  pathological 
standpoint,  though  it  plays  an  important  role  in  secondary  infec- 
tion in  phthisis  and  bronchiectasis. 


COCCUS  OF  MALTA  FEVER 

Micrococcus  Melitensis. 

Bacterium  Melitensis. 

Bacillus  of  Malta  Fever. 

Coccus  of  Malta  Fever. 

An  organism  belonging  somewhere  between  the  Coccacae  and 
Bacteriacae.  It  is  small,  oval-shaped,  and  of  about  .$n  diameter, 
occurring  in  culture  singly,  in  pairs,  or  in  chains.  In  the  latter 
form,  the  organism  elongates  and  resembles,  more  strongly,  bacilli. 
It  is  non-motile  and  it  has  no  spores.  Stain  faintly  with  the  com- 
mon basic  dyes,  but  not  by  Gram's  method.  It  has  been  found  in 
the  blood  during  life,  and  by  splenic  puncture. 

Cultures. — On  gelatine  its  growth  is  slow,  without  liquefaction. 
On  agar  the  growth,  at  37°C.,  is  more  rapid.  The  colonies  are 
pearly  white,  becoming  yellow.  In  bouillon  it  produces  turbidity, 
with  a  flocculent  deposit.  No  pellicle  is  formed.  On  potato  an 
invisible  growth  occurs.  Milk  is  not  coagulated,  nor  are  acids  or 
gases  produced. 

Pathogenesis. — It  causes  in  man,  Malta  fever.  Rabbits, 
guinea  pigs,  and  mice  are  not  susceptible  to  inoculation,  but  the 
disease  can  be  produced  in  monkeys. 

Agglutination. — The  serum  from  an  individual  suffering  from 
Malta  fever  agglutinates  the  bacilli,  even  in  dilutions  as  high  as  j 
i-ioo. 


INFLUENZA  BACILLUS  159 

Diagnosis  of  the  disease  can  be  effected  by  the  agglutination 
test,  and  by  splenic  puncture,  and  blood  cultures. 

It  is  present  in  the  blood  and  is  excreted  via  the  urine  and  milk. 
The  goat  while  not  suffering  with  Malta  fever  can  carry  the  germs 
in  its  body  and  excrete  them  in  the  milk.  Goats'  milk  is  a  general 
food  in  Malta.  The  inference  is  obvious.  Flies  may  transmit  the 
bacilli. 

INFLUENZA  BACILLUS 

Bacterium  Influenzse. 

Influenza  bacillus. 

Morphology  and  Stains. — Very  small  short  rods  which  are 
often  in  pairs,  found  within  epithelial  and  pus  cells,  and  in  sputum; 
from  40  to  80  in  a  cell.  May  grow  out  into  short  mycelia.  No 
flagella  or  spores  are  formed.  Stains  weakly.  Carbol-fuchsin,  di- 
luted, gives  the  best  result.  The  ends  of  the  bacillus  stain  more 
deeply  than  do  the  rest  of  the  cell.  It  is  decolorized  by  Gram's 
stain. 

Oxygen  Requirements. — It  is  a  strict  aerobe. 

Cultures  grow  best  on  blood-smeared  agar,  or  in  blood  bouillon 
between  27°  and  4i°C.;  best  at  37°C.  Blood  or  haemoglobin  is 
demanded  for  all  cultures.  In  bouillon  it  grows  in  thin  white 
flocculi.  On  agar  in  small  transparent  "dewdrop"  colonies, 
never  luxuriantly.  Grown  in  the  same  culture  with  Staphylo- 
coccus  aureus,  it  increases  more  luxuriantly  (symbiosis).  It  is 
probable  that  the  cocci,  in  some  way,  alter  the  blood  of  the  culture 
media.  Very  satisfactory  media  may  be  made  by  heating  blood 
agar  or  by  the  addition  of  sodium  oleate  to  it. 

Vitality. — It  is  easily  killed  by  light,  heat  and  drying.  Lives 
but  a  day  in  distilled  water,  and  from  eight  to  twenty-four  hours 
in  dried  sputum. 

Habitat. — Never  outside  the  body;  always  a  strict  parasite.  It 
is  found  in  the  mucous  membranes  of  the  upper  respiratory  tract, 
and  in  the  mucous  secretions. 


l6o  BACTERIA 

Pathogenesis. — Catarrhal  symptoms  follow  the  smearing  of  a 
culture  upon  the  nasal  mucosa  of  monkeys.  Pure  cultures, 
injected  into  the  peritoneum  of  guinea  pigs  cause  fatal  peritonitis. 
This  bacillus  was  isolated  by  Pf eiffer  during  the  influenza  epidemic 
of  1889  and  by  him  believed  to  be  the  cause  of  the  disease.  Be- 
tween that  time  and  the  pandemic  of  1918  it  has  been  found  in 
acute  respiratory  infections,  chronic  bronchitis,  sinusitis,  otitis  and 
meningitis,  all  attacks  being  characterized  by  great  depression. 
Conviction  has  never  been  obtained  that  it  was  the  principal  cause 
of  acute  disease  except  for  meningitis,  but  the  bronchitis  of  tuber- 
culosis has  been  ascribed  to  it  on  many  occasions.  The  pandemic 
cases  of  1918  showed  a  high  percentage  of  positive  findings  in  the 
sputum  and  lungs  at  autopsy.  There  was  however  no  definite  in- 
crease in  agglutinins,  lysins  or  complement  fixing  anti-bodies  so  that 
many  have  doubted  its  etiological  relation  in  epidemic  influenza.  It 
was  present  in  a  large  percentage  of  cases  and  certainly  aggravated 
pneumonitis  and  sinusitis  in  association  with  streptococci.  Its 
effect  seems  due  to  an  endotoxin,  possibly  also  to  some  exotoxin, 
having  a  definite  affinity  for  the  nervous  system.  It  circulates 
in  the  blood  rarely,  principally  in  the  meningitic  form  and  in 
early  stages  of  the  intense  general  cases. 

Influenzal  meningitis  is  more  frequent  than  formerly  or 
at  least  is  more  often  diagnosed.  It  can  be  reproduced  in 
monkeys. 

By  immunizing  a  goat  with  influenza  bacilli  Wollstein  obtained 
a  serum  which  has  a  pronouncedly  favorable  effect  upon  the 
experimental  disease  in  monkeys  and  promises  some  therapeutic 
power  for  human  beings.  Its  most  important  effect  is  to  stimu- 
late phagocytosis  in  the  cerebro-spinal  fluid. 

A  short  immunity  remains  after  a  spontaneous  attack  in  man 
but  attempts  at  production  of  immunity  by  vaccines  have  been 
disappointing.  By  the  use  of  a  mixed  vaccine  of  influenza 
bacilli,  streptococci  and  pneumococci,  the  chance  of  pneumonic 
complications  seems  reduced,  but  influenza  may  not  be  prevented. 


INFLUENZA  BACILLUS  l6l 

Therapeutic  use  of  vaccines  is  useful  only  to  increase  leucocytes 
which  are  characteristically  low  during  the  disease. 

Bordet-Gengou  Bacillus  of  Whooping  Cough. — This  is  a  very 
minute  ovoid  rod  lying  separately,  varying  from  .8-1.5^  long  and 
being  .^fj,  wide.  No  spores,  no  motility  or  flagella.  Stains  poorly, 
best  at  ends;  Gram-negative.  It  may  be  cultivated  from  expec- 
toration early  in  the  disease  upon  media  containing  glycerine, 
potato,  blood  and  agar.  Aerobe,  and  grows  best  at  37°C.  There 
is  an  endo-toxin.  Infective  for  monkeys.  The  discoverers  claim 
this  to  be  the  cause  of  pertussis,  because  it  will  act  as  an  antigen 
and  fix  complement  away  from  the  hemolytic  series. 

Its  relation  to  pertussis  has  been  explained  by  the  report  that  it 
is  found  lying  between  the  cilia  of  the  respiratory  epithelium,  an 
embarrassment  of  the  movements  of  which  causes  the  coughing 
attack.  The  only  bacteriological  diagnosis  is  by  growing  the 
microbes  on  the  medium  given  above,  and  by  the  complement 
fixation  test.  Vaccines  are  said  to  have  a  prophylactic  value, 
and  some  relief  of  paroxysms  certainly  seems  to  follow  their  use. 

The  last  two  microorganisms  are  types  of  the  so-called  hemo- 
globinophilic  bacteria  because  of  the  requirement  of  blood 
coloring  matter  in  laboratory  culture  media.  Other  forms  have 
been  found  in  trachoma  and  in  the  spinal  fluid. 

Conjunctivitis. — There  are  two  specific  germs  for  conjunctivitis 
separate  from  the  gonococcus.  They  are  the  bacillus  of  Koch- 
Weeks  and  that  of  Morax  and  Axenfeld. 

Koch-Weeks  Bacillus. — The  organism  of  pink  eye.  This  is 
a  minute,  i.5juX.2/-i  non-motile,  Gram-negative,  sporeless,  poorly 
staining  rod,  very  like  the  influenza  bacillus.  It  is  aerobic  and 
non-liquefying.  It  grows  as  minute,  pearly,  glistening,  discrete 
colonies  only  upon  agar  of  5  percent  strength  plus  serum. 

The  Bacillus  of  Morax  and  Axenfeld. — A  non-motile,  sporeless 
diplo-rod;  negative  to  Gram  stain.     Grows  only  in  the  presence  of 
serum  or  blood  and  liquefies  the  former.     It  is  larger  than  the 
Koch- Weeks  bacillus,  measuring  up  to  2/*. 
11 


l62 


BACTERIA 

PLAGUE  BACILLUS 


Bacterium  Pestis. 

Plague  Bacillus  (Fig.  42). 

Morphology  and  Stains. — Short  plump  rods  with  rounded  ends, 
containing  no  spores  and  non-motile.  It  is  said  by  some  that  a 
capsule  is  formed.  Organisms  from  exudates,  or  blood,  exhibit 
characteristically  peculiar  polar  staining.  They  are  often  found 


FIG.  42. — Pest  Bacilli  from  spleen  of  rat.     (Kolle  and  Wassermann.) 

within  the  leucocytes.  In  bouillon  the  organism  grows  in  long 
chains;  is  stained  with  all  the  common  basic  dyes,  but  is  not 
colored  by  Gram's  method  in  cultures.  It  exhibits  a  great  variety 
of  involution  forms  when  grown  in  salty  culture  media  (3^ 
percent  salt). 

Relation  to  Oxygen. — Strict  aerobe,  the  growth  is  stopped  by 
the  exclusion  of  oxygen. 

Vital  Requirements.— Grows  well  at  22°C.,  but  best  at  35°C.; 
is  killed  after  a  short  exposure  to  55°-6o°C.,  stands  drying  from 
four  to  eight  days,  and  dies  in  water  after  a  week.  In  the  buried 
bodies  of  man  and  animals  it  lives  from  twenty-two  to  thirty-eight 
clays.  Withstands  freezing  for  months,  but  does  not  stand  light 
or  chemicals  very  long. 


PLAGUE  BACILLUS  163 

Cultures. — Grows  very  well  on  culture  media.  In  bouillon  it 
thrives  abundantly,  with  a  heavy  pellicle  which  produces  dependent 
stalactites  that  drop  to  the  bottom  of  the  vessel.  On  gelatine 
plates  it  grows  in  small  flat  colonies,  which  are  gray  and  trans- 
parent, and  which  do  not  liquefy  the  gelatine  (Fig.  43).  In  gela- 
tine tubes  it  forms  a  faint  thread-like  line,  without  liquefying 
the  media.  On  agar  the  growth  is  whitish  and  abundant,  and 
resembles  the  colon  bacillus.  Old  cultures  are  luxuriant.  Milk 


FIG.  43. — Colonies  of  plague   bacilli   forty-eight  hours   old.     (Kolle   and 

(Wassermann.) 

is  not  coagulated,  but  a  faint  acidity  appears.  Potato  yields  a 
slow  whitish-yellow  growth  that  is  sharply  outlined. 

Chemical  Activities. — Does  not  produce  H^S,  enzyme,  colors, 
or  odors,  indol  or  nitrites.  The  toxin  produced  is  not  soluble  and 
the  nitrate  is  non-poisonous.  Old  killed  bouillon  cultures  can 
be  extracted  and  a  highly  poisonous  substance  precipitated  there- 
from with  alcohol,  or  ammonium  sulphate,  that  is  lethal  for  mice. 

Habitat. — Never  found  in  healthy  human  bodies.  In  persons 
afflicted  with  plague,  the  organism  is  widely  distributed  in  buboes 
and  in  the  cutaneous  pustules,  lymphatics  and  in  the  lungs  in 
plague  pneumonia;  more  rarely  in  the  blood  and  other  organs. 


164  BACTERIA 

In  animals,  plague  occurs  in  rats.  It  is  supposed  that  some 
tropical  soil  bacilli  infect  rats,  and  becoming  accustomed  to  the 
rodent's  body,  are  eventually  transmitted  to  man.  The  bacilli 
may  be  transmitted  from  rat  to  rat  in  India  by  the  rat  fleas 
which  also  can  bite  man.  The  organisms  remain  in  the  flea  for 
some  time.  Rats  are  also  infected  from  dead  rats.  In  epidemic 
times  the  soil  becomes  infected  and  persons  going  barefoot  may 
be  infected. 


FIG.  44. — B.  Pestis  in  pus  of  bubo.   .  (Jackson.) 

Pathogenesis. — Highly  pathogenic  for  man.  Is  the  cause  of 
the  bubonic  or  Oriental  plague;  bacilli  gain  entrance  by  way  of 
the  skin,  causing  localized  foci  of  infection  from  which  buboes 
develop,  followed  by  pest-sepsis  and  death.  The  lungs  may  be 
the  original  site  of  invasion,  and  plague  pneumonia  (worst  form 
of  the  disease)  may  result.  The  typical  bacilli. can  be  found  in 
the  sputum  of  the  patient  thus  affected  but  not  in  quietly  expired 
air.  The  mortality  from  this  plague  is  from  50  percent  to  80 
percent. 

Almost  all  domestic  animals — rats,  mice,  guinea  pigs,  rabbits 
and  squirrels  are  susceptible;  horses  and  swine  are  very  suscep- 
tible; cows  and  dogs  less  so.  Rats  seem  to  be  affected  with  a 


PLAGUE  BACILLUS  165 

chronic  form  of  the  malady,  and  by  inhabiting  ships  and  ware- 
houses in  foreign  countries,  spread  the  disease.  Post  mortems 
on  infected  animals  reveal  haemorrhagic  petechia  and  serous 
infiltration  into  serous  cavities.  Death  is  generally  due  to  a 
profound  toxaemia  and  exhaustion. 

The  virulence  of  the  organism  can  be  raised  by  passing  it 
through  a  series  of  animals. 

Serum  from  infected  animals  agglutinates  plague  bacilli. 


FIG.  45. — Pest  bacillus  involution  forms  produced  by  growing  on  3  percent 
salt  agar.     (Kolle  and  Wassermann.) 

The  diagnosis  of  the  plague  bacilli  is  made  by  rubbing  the  sus- 
pected culture  upon  the  freshly  shaven  skin  of  a  guinea  pig;  if 
the  animal  develops  buboes  and  dies,  and  polar  staining  bacilli 
are  found,  it  is  probable  that  the  organism  is  the  plague  bacillus. 
Further,  if  curious  involution  forms  develop  an  heavily  salted 
agar  (3  percent)  the  diagnosis  is  confirmed  (Fig.  45). 

Immunity. — It  is  possible  to  immunize  against  the  disease. 
Kitasato  and  Yersin  produced  an  anti-toxic  serum,  which  has, 
not  only  a  prophylactic,  but  a  curative  action.  By  the  use  of 
killed  culture  Haffkine  vaccinated  many  people  against  the  plague 
very  successfully  (see  page  83). 


I 66  BACTERIA 

MUCOSUS  CAPSULATUS  GROUP 

There  is  a  large  group  of  organisms  of  moderate  pathogenic 
powers  and  importance  called  variously,  Bacterium  aerogenes, 
Bacterium  mucosus  or  Aerogenes  mucosus  group  of  which  the 
Friedlander  bacillus  is  the  most  important.  They  all  have  a 
luxuriant  growth  on  media;  are  negative  to  Gram  stain;  ferment 
most  of  the  carbohydrates;  are  non-motile  and  most  of  them 
show  a  capsule  when  in  the  animal  body. 

Perkins  divides  them  as  follows: 

I.  Bacterium  aerogenes  type  ferments  all  carbohydrates  with 
gas. 

II.  Bacterium  pneumoniae  group  ferment  all  carbohydrates  but 
lactose,  with  gas. 

III.  Bacterium  lactis  aerogenes  group  ferment  all  carbohydrates 
except  saccharose,  with  gas. 

These  organisms  are  important  members  of  the  intestinal  flora. 

Bacterium  Pneumoniae. 

Friedlander 's  Pneumonia  Bacillus. 

Morphology  and  Stains. — Short  plump  rods  with  rounded  ends, 
surrounded  by  a  thick  gelatinous  capsule  .in  animal  fluids,  and 
when  grown  in  milk;  is  not  motile,  and  has  no  spores;  does  not 
stain  by  Gram's  method,  but  easily  by  the  common  basic  dyes. 

Oxygen  Requirements. — Grows  in  and  without  oxygen,  upon 
all  culture  media. 

Chemical  Activities. — Produces  abundant  acids,  COz  and  H, 
gas,  alcohol,  indol,  ferment  and  H2S. 

Habitat. — Has  been  found  in  soil;  sometimes  in  healthy  saliva. 

Culture  Media. — Grows  luxuriantly  on  all  culture  media. 

On  gelatine  it  grows  in  roundish  elevated  colonies  that  are 
yellowish-white  with  a  slimy  lustre,  and  never  liquefies  the  gela- 
tine. In  agar  it  multiplies  even  more  abundantly  with  a  moister 
growth.  The  border  of  streak  cultures  is  smooth  and  wavy,  and 
the  water  of  condensation  is  cloudy.  In  bouillon  the  growth  is 


MUCOSUS   CAPSULATUS   GROUP  167 

very  cloudy  with  a  silvery  deposit  at  the  bottom.  The  bouillon 
becomes  thickened.  Milk  is  not  coagulated,  and  potato  yields 
a  luxuriant  yellowish,  moist  shining  growth. 

Pathogenesis. — It  is  possible  to  cause  pneumonia  in  mice,  also 
septicaemia.  Guinea  pigs  and  dogs  are  susceptible.  It  may  be 
found  in  normal  mouths.  Friedlander's  pneumonia  is  much  less 
frequent  than  that  due  to  the  pneumococcus,  but  it  is  very  fatal. 
Members  of  this  group  may  also  be  responsible  for  cystitis, 
pyelitis,  sinusitis  and  in  children  pneumonia  and  pleuritis.  The 
exudate  produced  by  all  of  them  is  mucoid,  stringy.  Anti-bodies 
are  not  readily  formed  against  any  of  the  mucosum  group  so  that 
anti-sera  and  vaccines  are  not  of  great  value. 

The  Bacterium  lactis  aerogenes  group  is  a  very  large  one  and 
includes  nearly  all  the  forms  engaged  in  milk  souring.  The 
ordinary  B.  lactici  is  very  like  the  colon  bacillus,  but  is  non-motile. 
It  forms  lactic  acid  among  its  principal  products.  The  most 
important  lactic  acid  producer  related  to  but  not  belonging 
directly  in  this  group,  is  Bact.  bulgaricum  of  Massol.  This  is  the 
principal  ferment  of  the  eastern  sour  milks,  Kumyss  and  Yoghurt. 
Because  of  the  large  amount  of  lactic  acid  formed  by  this  germ, 
Metchnikoff  has  advocated  cultures  of  it  and  sour  milk  made 
by  it  in  the  treatment  of  intestinal  putrefaction  and  fermentation. 
The  Bacterium  bulgari^um  produces  a  soft  milk  curd  and  an  excess 
of  lactic  acid  and  alcohol.  The  bacteria  are  non-motile,  non- 
spore-forming,  Gram-positive  and  vary  from  21*  to  5oju  in  length. 
They  grow  with  difficulty  in  the  laboratory,  best  on  milk  and 
whey.  Optimum  temperature  44°C.  They  form  branching  fila- 
mentous colonies.  Milk  is  coagulated  in  eighteen  hours  at  44°C. 
and  in  thirty-six  hours  at  37°C.  The  clot  is  not  dissolved.  Gela- 
tine is  not  liquefied.  Congeners  with  this  organism  are  Bac. 
acidophilus  and  Bac.  acidophil-aerogenes  differing  from  it  in  fer- 
mentative powers. 


1 


1  68  BACTERIA 

TYPHOID  BACILLUS 

Bacterium  Typhi.    Eberth. 

Bacillus  Typhosus 

Typhoid  Bacillus  (Fig.  46). 

A  most  important  pathogenic  organism  which  causes  typhoid 
fever. 

Morphology  and  Stains.  —  Generally  short  plump  rods  i  to  3/4 
long,  and  .6  to  .8/x  broad.  Forms  long  threads  in  cultures, 
especially  on  potatoes.  Polar  metachromatic 
bodies  are  sometimes  seen  as  are  unstained 
areas  when  alkaline  methylene  blue  is  used. 
The  rod  is  flagellated  (peritrichous)  ;  con- 
tains no  spores;  exhibits  pleomorphic  and 
involution  forms;  is  actively  motile,  and 
stains  with  all  the  basic  aniline  dyes,  but 


ella.     (Kolle    and       Vital    Resistance.—  The    thermal    death- 

Wassermann.)  point  ig  6Qoc  ^   ten  tQ  fifteen  minutes>      Re_ 

mains  alive  in  ice  for  three  months;  even  the  temperature  of 
liquid  air  does  not  destroy  it  at  once.  In  distilled  water  it  lives 
for  months,  but  if  other  saprophytic  bacteria  are  associated 
with  it,  however,  it  quickly  dies.  Does  not  resist  drying  or 
chemicals,  except  carbolic  acid,  towards  which  it  exhibits  a 
tolerance.  Sunlight  kills  it  in  an  hour. 

Habitat.  —  It  never  exists  in  nature,  except  where  water  or  soil 
has  been  contaminated  by  fasces  or  urine.  It  may  multiply  in 
potable  waters,  in  milk,  and  the  juices  of  oysters. 

Chemical  Activities.  —  Does  not  produce  proteolytic  enzymes; 
forms  H2S,  but  will  not  ferment  the  sugars  with  gas  formation. 
Does  not  yield  indol  or  nitrites.  Produces  levorotatory  lactic 
acid.  Its  toxin  is  all  contained  within  the  bacterial  cell  (endo- 
toxins)  and  is  not  water-soluble.  This  toxin  is  manifested  by 
injecting  washed  and  killed  bacilli  into  animals,  or  by  freezing  the 


TYPHOID  BACILLUS 


169 


bacilli  with  liquid  air,  and  then  crushing  them.  This  injected 
into  guinea  pigs  causes  diarrhoea,  mydriasis  and  death. 

Oxygen  Requirements. — It  is  a  facultative  aerobe. 

Cultural  Characteristics. — It  grows  upon  all  media  at  the  tem- 
perature of  the  body,  37°C.  and  more  slowly  at  2o°C.  On  gela- 
tine plate  it  produces  at  first  small  colonies,  yellowish  and  punctate, 
which  become  whitish,  delicately  notched  and  ridged  (Fig. 
47).  In  gelatine  stab  culture  it  grows  in  a  thread-like  granular 


FIG.  47. — Seventy-two  hour  old  culture  of  typhoid  bacillus  on  gelatine. 
(Kolle  and  Wassermann.) 

line,  without  producing  gas.  In  neither  case  is  the  gelatine  lique- 
fied. On  agar  plates  the  colonies  are  not  so  characteristic,  being 
round,  grayish-white,  and  shining.  In  milk  it  grows  well,  not 
coagulating  it  even  after  boiling,  and  only  a  very  little  acid  is 
produced.  On  acid  potato  the  growth  is  characterized  by  its 
invisibility,  and  this  fact  is  used  to  differentiate  it  from  other 
kindred  bacteria.  The  growth  is  detected  only  by  scratching 
with  a  needle.  In  bouillon  it  grows  uniformly,  producing  very 
little  acid,  and  no  gas.  In  special  media  (Hiss's  semi-solid  media) 
thread-like  colonies  are  produced,  which  are  characteristic.  On 
Eisner's  potato  media  it  produces  small  granular,  glistening 


1 70  BACTERIA 

points.  It  also  grows  characteristically  in  Endo  and  the  Drigalski 
and  Conradi  media.  In  sugar  media  no  gas  is  formed  but  there 
is  acidification  in  dextrose,  galactose,  mannit,  maltose,  xylose  and 
levulose.  The  addition  of  sterile  bile  to  culture  medium,  10-50 
percent,  increases  the  chance  of  isolating  the  germ  in  blood,  faeces 
or  urine  cultures;  it  acts  by  inhibiting  other  organisms  and  by 
supplying  salts  favorable  to  the  typhoid  bacillus. 

Invasion  of  Body. — This  organism  generally  invades  the  body 
by  way  of  the  alimentary  tract,  in  food  and  water.  Flies  may 
infect  milk  and  other  foods.  Oysters  may  become  infected  and 
cause  disease.  Personal  contact,  by  hand  to  hand  for  example, 
is  a  very  potent  method  of  transmission. 

Pathogenesis. — It  is  certainly  the  cause  of  typhoid  fever. 
During  the  attack  the  germs  circulate  in  the  blood-stream  during 
the  entire  fastigium  but  are  obtained  with  ease  only  in  the  first 
two  weeks.  They  are  constantly  in  the  faeces  for  varying  periods 
even  after  clinical  recovery  and  may  be  frequently  found  in  the 
urine.  Rose  spots  also  contain  them.  Also  found  in  the  spleen 
and  gall-bladder.  It  produces  well-marked  histological  changes 
in  the  lymphoid  structures,  particularly  in  Peyer's  patches, 
solitary  foUicles,  and  other  lymph-glands.  There  is,  according 
to  Mallory,  a  massive  endothelial  proliferation  in  the  lymph- 
glands.  This  causes  occlusion  of  the  lymph-vessels,  and  is  fol- 
lowed by  necrosis  (ulceration)  of  the  Peyer's  patches.  The  in- 
tense phagocytic  action  of  the  fixed  lymphatic  cells  in  the  glands 
is  manifest  toward  the  red  blood  cells,  which  are  devoured  in 
great , numbers.  The  toxin  causes  degeneration  of  other  organs, 
particularly  in  the  liver.  Bacilli  are  found  in  the  spleen  and 
blood.  The  rose«-colored  spots  are  found  to  be  full  of  them. 
The  disease  is  certainly  not  a  merely  localized  infection  of  the 
lymph  structures,  but  is  a  bacteriaemia.  There  is  often  a  mixed 
infection  in  which  streptococcus  pyogenes  in  the  blood  plays  an 
active  role.  In  the  necrosis  of  bone  and  in  subphrenic  abscess 
the  typhoid  bacilli  may  act  as  a  pus  former.  Commonly  it 


TYPHOID  BACILLUS 


171 


produces  death  by  (i)  profound  toxaemia;  (2)  ulceration  of  the 
Peyer's  patches,  causing  perforation  and  peritonitis;  (3)  by  the 
destruction  of  a  blood-vessel  in  the  floor  of  an  ulcer  producing  a 
haemorrhage. 

In  animals,  as  a  rule,  typhoid  bacilli  if  injected,  produce  no 
disease,  and  the  bacilli  rapidly  die.  In  chimpanzees,  how- 
ever, it  is  possible  to  produce  atypical  typhoid  lesions  and 
symptoms. 

Natural  and  Acquired  Immunity. — Human  blood  serum  is 
strongly  bactericidal  toward  the  typhoid  bacillus.  Normal  gas- 
tric juice,  with  its  hydrochloric  acid,  destroys 
the  bacillus  when  ingested  and  this  forms  the 
natural  means  of  protection.  Immunity  fol- 
lowing an  attack  of  typhoid  is  generally  of 
long  duration.  If  bacilli  do  reach  the  blood- 
stream of  an  immune  individual,  the  ambo- 
ceptors  originated  by  a  previous  infection, 
together  with  the  complement  normally  pres- 
ent, effect  a  solution  of  the  invading  organism. 
For  vaccination  against  typhoid  fever  see 
page  80.  Anti-serum  for  typhoid  has  been 
prepared  by  injecting  horses  with  killed 
culture  of  typhoid  bacilli,  but  it  has  not 
proved  to  be  effective. 

Agglutinations. — One  of  the  most  important  means  of  diagnos- 
ing typhoid  fever  is  by  the  so-called  Widal  test,  really  the  Gruber 
and  Durham  agglutination  reaction.  This  consists  in  applying 
the  serum  of  the  blood  of  a  person,  supposedly  ill  with  typhoid, 
to  a  fresh  bouillon  culture  of  typhoid  bacilli.  If  the  person  has 
the  disease,  and  it  has  lasted  for  five  or  more  days,  the  bacilli  are 
promptly  agglutinated  in  clumps.  Undiluted  normal  serum,  and 
serum  from  people  suffering  other  diseases,  will  bring  about  the 
same  reaction  at  times;  it  is  therefore  best  to  dilute  the  serum 
with  water  1-50,  and  if  the  reaction  comes  within  an  hour  the 


FIG.  48— Widal 
reaction.  One-half 
of  the  field  shows 
typhoid  bacilli  un- 
clumped,  other  half 
shows  clumping. 
(Greene's  Medical 
Diagnosis.) 


172  BACTERIA 

disease  is  considered  typhoid  fever.  The  test  may  be  either  with 
a  hanging  drop  and  examined  microscopically,  or  macroscopically 
by  adding  a  drop  of  diluted  serum  to  fresh  bouillon  culture  of 
typhoid  bacilli,  when,  if  the  case  is  typhoid,  large  clumps  of  the 
bacilli  will  form  and  drop  to  the  bottom  of  the  tube.  Animals 
immunized  against  typhoid  exhibit  this  reaction  to  a  high  degree. 
Serum  diluted  with  10,000  parts  of  water  has  caused  the  reaction 
in  less  than  one  hour's  time.  This  reaction  with  a  known  culture 
of  typhoid  bacilli  is  used  clinically  to  identify  serum  from  a  doubt- 
ful case  of  typhoid,  and  establish  a  diagnosis.  On  the  other  hand, 
a  known  serum  prepared  artificially  by  immunizing  rabbits  with 
bacilli  is  used  to  identify  typhoid  bacilli  when  found  in  water, 
or  elsewhere.  There  are  two  stages  to  the  reaction;  immediately 
after  mixing  the  serum  and  culture,  the  bacilli  will  be  seen  to 
become  less  motile,  and  then  still.  After  this  they  begin  to 
huddle  together  into  clumps.  In  complete  reaction  they  remain 
immobile  and  tightly  massed.  In  some  cases  bacteriolysis  occurs, 
and  many  of  the  bacteria  are  dissolved  in  the  serum.  The  foetus 
of  a  woman  suffering  from  typhoid  contains  agglutinins  in  its 
blood.  The  milk,  tears,  and  other  body  fluids  from  an  individial 
with  typhoid,  agglutinate  typhoid  bacilli.  Serum  to  perform  the 
test  may  be  obtained  by  puncturing  the  skin,  or  by  blistering  it 
and  drawing  off  the  serum,  or  else  by  abstracting  blood  from  a 
vein  with  a  hypodermic  syringe. 

Agglutinin  appears  during  typhoid,  generally  after  the  fifth  day, 
and  persists  for  some  time  (several  years?)  after  convalescence,  01 
if  the  actual  agglutinin  titer  be  not  high  in  an  immune  person,  il 
rises  rapidly  should  typhoid  bacilli  gain  entrance  to  the  body. 
In  persons  vaccinated  against  the  germ,  the  agglutinin  titei 
remains  partly  high  for  at  least  six  months  but  when  the  amounl 
shall  have  returned  to  normal,  it  has  the  power  to  revive  rapidb 
should   infection   threaten.     Agglutinins   are  in   a  measure  ai 
index  of  resistance,  or  at  least  their  facility  of  action  is  01 
guarantee  of  protection. 


PARATYPHOID  BACILLUS  173 

Paratyphoid  Bacillus. — A  pathogenic  organism  producing  all 
the  clinical  symptoms  of  typhoid,  only  in  milder  form  (at  times) 
has  been  discovered.  It  differs  from  the  true  bacillus  because  it 
ferments  dextrose  and  maltose  producing  gas  and  acid,  and  is  not 
agglutinated  by  the  serum  from  a  true  typhoidal  infection. 
There  are  several  closely  related  varieties  differing  in  growth  upon 
litmus  milk  and  in  fermenting  several  sugars  but  in  other  respects 
they  resemble  the  typhoid  bacillus,  and  seem  to  occupy  a  position 
between  it  and  the  colon  bacillus.  Paratyphoid  endotoxin  resists 
6o°C.  from  thirty  to  sixty  minutes  and  in  the  case  of  the  organisms 
of  meat  poisoning,  paratyphoid  beta,  paracolon  and  the  Gartner 
bacillus,  a  short  exposure  to  the  boiling  point  does  not  seem  to 
destroy  the  toxin. 

It  is  generally  taught  today  that  the  foregoing  organisms 
produce  infections  of  similar  clinical  characters  in  that  they  are 
contracted  in  the  same  manner,  have  comparable  pathology 
and  immunity  reactions  and  are  amenable  to  the  same  pro- 
phylactic measures;  they  are  designated  "the  typhoid  fevers." 

The  Paracolons  are  organisms  like  the  paratyphoids,  but  some- 
what closer  to  the  colon  bacillus  (for  example,  see  page  177). 
This  term  is  best  applied  to  organisms  of  the  meat  poisoning 
group,  as  the  Gartner  bacillus,  the  hog  cholera  bacillus,  so  that 
the  varieties  which  cause  typhoid  fever  in  man  can  be  recognized 
under  the  term  paratyphoid. 

Blood  cultures  are  often  employed  in  large  hospitals  for  the 
diagnosis  of  typhoid  fever.  During  the  first  week  of  the  attack 
bacilli  may  be  recovered  from  the  blood  by  withdrawing  10  c.c. 
of  blood  from  a  vein  and  mixing  it  with  500  c.c.  of  bouillon.  The 
large  amount  of  blood  is  necessary,  because  the  bacilli  are  few  in 
number,  and  the  bactericidal  action  of  the  serum  outside  the 
body  is  powerful  until  mixed  with  the  bouillon,  after  which  the 
bacilli  are  able  to  withstand  it.  The  bacilli  may  be  easily  isolated 
from  the  blood  by  adding  the  latter  to  some  bile  and  then  incubat- 
ing it.  From  the  bile,  cultures  are  made  in  agar  or  in  bouillon. 


1 74  BACTERIA 

COLON  BACILLUS 

Bacterium  Coli. 

Bacillus  coll  or  Bacillus  coll  communis. 

Colon  Bacillus. 

While  not  strictly  a  pathogenic  organism,  it  plays  such  an  im- 
portant part  in  secondary  infection,  and  resembles  so  closely  the 
typhoid  bacillus,  that  it  will  be  described  here. 

Morphology  and  Stains. — Is  not  so  motile  as  typhoid;  has  not 
so  many  flagella;  and  is  devoid  of  spores.  It  exhibits  pleomor- 
phism;  may  grow  in  chains;  and  possesses  vacuoles  and  polar 


FIG.  49. — Colon  bacillus  showing  flagella.     (Kolle  and  Wassermann.) 

bodies  at  times.  Is  readily  stained  by  all  the  common  basic 
stains,  but  not  by  Gram's  method. 

Oxygen  Requirements. — It  grows  especially  well  in  oxygen; 
without  oxygen  its  growth  is  not  so  good. 

Temperature  requirements,  and  vital  resistance.  It  grows  well 
at  room  and  incubator  temperature.  Its  thermal  death-point  is 
about  62°C.;  light  and  heat  are  destructive  to  it,  and  its  resis- 
tance to  antiseptics  is  somewhat  less  than  that  of  typhoid  bacillus. 


COLON  BACILLUS  175 

Cultures. — Thrives  in  all  common  culture  media,  especially  if 
sugar  is  present.  It  is  restrained  by  excess  of  acids  produced  in 
culture  media.  On  gelatine  it  grows  like  the  typhoid  bacillus 
(from  which  it  is  difficult  to  differentiate,  see  page  176)  in  whitish 
raised  colonies  that  do  not  liquefy  the  media.  Sometimes  the 
growth  is  thin  and  iridescent,  and  exhibits  bizarre  shapes — 
tadpole-like  and  lobulated.  Typhoid  colonies  show  deep  furrow- 
like  ridges  under  the  microscope.  In  the  special  semi-solid  media 
of  Hiss,  the  typhoid  produces  uniform  cloudiness,  with  thread- 
like colonies.  The  colon  does  not  so  quickly  cause  this  cloudi- 
ness, and  forms  gas  bubbles.  On  agar  plates  surface  colonies  are 
like  typhoid,  only  they  are  thicker  and  moister.  If  litmus  is 
added  to  this  medium,  a  red  zone  forms  about  the  colonies  due 
to  the  presence  of  lactic  acid.  In  agar  tubes  the  growth  is  more 
luxuriant  and  resembles  typhoid.  In  litmus  bouillon  it  rapidly 
reddens  the  litmus,  clouds  the  medium,  and  deposits  a  slimy 
sediment.  In  milk  it  always  produces  coagulation.  On  potato 
it  grows  more  rapidly  and  luxuriantly  than  typhoid,  at  first 
yellowish-white,  which  later  changes  to  yellowish-brown.  It  is 
slimy. 

Chemical  Activities. — Produces  color  on  potato  only.  Sugars 
are  fermented  with  the  production  of  H,  COg  and  some  N. 
It  ferments  glucose,  lactose,  saccharose,  maltose,  dulcit  and 
some  others  with  the  production  of  gas.  Produces  lactic,  acetic 
and  formic  acids,  also  indol  abundantly,  and  H2S.  It  decomposes 
urea. 

Habitat. — Found  always  in  the  intestinal  contents  of  most  ani- 
mals and  man.  Also  in  streams  and  rivers  that  run  through  farm 
lands  and  by  towns.  While  it  is  difficult  to  find  typhoid  bacilli 
in  contaminated  drinking  water,  the  colon  bacilli  are  easily 
found.  If  in  abundance,  it  indicates  great  faecal  pollution.  In 
milk  it  is  often  found,  where  it  plays  an  important  part  in  souring. 

Pathogenesis. — It  is  pathogenic  to  rabbits  and  guinea  pigs, 
causing  peritonitis  if  injected  into  the  peritoneal  cavity.  In  man 


176  BACTERIA 

it  plays  rather  a  subordinate  pathogenic  role,  but  it  has  been 
found  the  causal  agent  of  some  cases  of  suppurative  appendicitis, 
peritonitis,  and  cystitis.  It  may  attack  the  lungs  and  meninges 
of  feeble  children,  and  cause  death  by  setting  up  a  pneumonia  or 
meningitis.  During  the  agonal  period  in  wasting  diseases  it  may 
cause  terminal  infection  and  hasten  death.  Colon  bacilli  encysted 
in  the  liver  and  kidney  have  been  found  by  Adami  in  cirrhosis  of 
these  organs,  and  it  is  believed  by  him  to  be  partly  the  cause  of 
these  diseases;  chronic  infections  of  the  rectum  are  due  to  this 
organism. 

Agglutination. — Animals  immunized  against  colon  bacilli  by 
repeated  injections,  exhibit  agglutinins  in  their  blood. 

The  differentiation  of  the  typhoid  from  the  colon  bacillus  is 
largely  accomplished  by  noting  the  chemical  reactions  of  both 
organisms  in  culture  media.  The  chief  differences  are : 

1.  The  typhoid  bacillus  has  more  flagella  than  the  colon,  and  is 
much  more  motile. 

2.  On  gelatine  culture  plates,  the  typhoid  colonies  develop 
more  slowly  than  the  colon,  and  are  much  more  delicate  and  trans- 
parent.    If  litmus  is  present  the  colon  colonies  are  red,   the 
typhoid  bluish. 

3.  In  media  containing  dextrose,  or  lactose,  gas  is  produced  by 
the  colon,  but  not  by  typhoid. 

4.  In  peptone  solution  the  colon  produces  indol,  while  the 
typhoid  does  not. 

5.  Milk  is  coagulated  by  the  colon,  but  not  by  the  typhoid. 

6.  Dn  potatoes  colon  grows  much  more  luxuriantly  than  typhoid. 

7.  Typhoid  reddens  neutral  red;  colon  changes  it  to  bright 
yellow. 

8.  The  m.ost  important  test  is  the  agglutinative  one.     Typhoid 
is  clumped  by  anti-typhoid  sera,  highly  diluted,  while  the  colon 
is  not. 

No  anti-sera  of  value  have  been  found  for  colon  bacillus  infec- 
tion, but  bacterins  have  been  used  with  much  benefit. 


GARTNER'S  BACILLUS  177 

GARTNER'S  BACILLUS 

Bacillus  Enteritidis. 

Bacillus  of  Gartner. 

The  cause  of  one  form  of  meat  poisoning,  and  closely  allied  to 
the  paratyphoid  bacillus  in  its  morphological  characteristics.  It 
gives  a  classical  picture  of  the  type  "paracolon." 

Morphology  and  Stains. — This  organism  is  a  short  plump 
ovoid;  is  motile;  has  about  eight  flagella;  does  not  form  spores; 
and  stains  well  with  all  the  common  aniline  dyes,  but  not  with 
Gram's  method. 

Vital  Resistance. — It  is  a  facultative  anaerobe.  It  is  destroyed 
by  means  outlined  for  the  colon  bacillus  when  in  culture.  In 
meat  it  must  be  subjected  to  prolonged  heating. 

Cultures. — Grows  on  all  the  common  culture  media.  In 
bouillon  thrives  well,  producing  gas  in  media  containing  dextrose. 
It  ferments  without  gas  production  lactose,  galactose,  maltose, 
and  cane  sugar.  Does  not  produce  indol,  which  distinguishes 
it  from  the  colon  bacillus,  to  which  it  is  closely  allied.  In  milk 
it  reduces  litmus  and  coagulates  the  casein  in  a  few  days.  On 
potato  it  grows  well,  producing  a  yellowish  shining  layer.  On 
gelatine  it  multiplies  without  liquefying  the  medium.  Super- 
ficial colonies  in  plates  are  pale  and  gray,  deep  colonies  yellow 
and  spherical. 

Chemical  Activities. — Acid,  gas  and  a  powerful  heat-resisting 
toxin  which  is  soluble,  are  found.  Infected  meat  contains  this 
toxin,  which  is  not  destroyed  by  cooking. 

Pathogenesis. — It  is  pathogenic  for  man,  horses,  cattle,  and 
laboratory  animals.  Neither  the  bacilli  nor  the  toxin  they  elabo- 
rate are  destroyed  by  moderate  heat.  Flesh  is  infected  before 
death,  after  which,  both  the  bacilli  and  toxin  increase.  Mischief 
follows  the  partaking  (usually  in  the  form  of  sausages,  etc.)  of 
this  meat,  causing,  in  men,  violent  nausea  and  diarrhoea,  skin 

eruption,  and  in  severe  cases,  pneumonia,  nephritis,  collapse  and 
12 


178  BACTERIA 

death.  Mortality  is  from  2  percent  to  15  percent.  The  post- 
mortem findings  are  not  specific.  There  may  be  evidence  of  an 
enteritis  with  swollen  lymph  follicles,  and  an  enlarged  spleen. 

Agglutination. — The  blood  of  infected  individuals  may  agglu- 
tinate bacilli.  A  dilution  of  such  blood  with  8,  ooo  parts  of  water 
has  produced  the  reaction. 

No  anti-serum  or  bacterin  treatment  is  as  yet  possible. 

DYSENTERY  BACILLUS 

Bacterium  Dysenteriae. 

Dysentery  Bacillus. 

The  cause  of  one  form  of  tropical  dysentery.  The  group  to 
which  this  belongs  comprises  many  closely  related  varieties  some 
of  which  are  thought  to  be  the  cause  of  infant  diarrhoea  in  this 
country.  There  are  numerous  varieties  of  this  organism,  the 
differentiation  of  which  depend  upon  their  chemical  activities, 
fermentation  of  various  carbohydrates  being  the  most  important, 
and  agglutinative  properties  with  different  sera.  The  tropi- 
cal form  of  dysentery  is  due  to  the  type  orginally  described  by 
Shiga;  this  type  is  uncommon  in  temperate  zones,  the  Flexner 
variety  being  much  more  common.  The  Shiga  variety  is  much 
the  more  virulent. 

Morphology  and  Stains. — The  organism  is,  in  many  respects, 
similiar  to  the  typhoid  bacillus,  but  is  plumper.  It  is  non-motile, 
has  no  spores,  and  exhibits  pleomorphism.  It  stains  well  with 
the  common  aniline  dyes,  but  not  by  Gram's  method. 

Vital  Properties. — It  is  killed  by  i  percent  carbolic  solution 
in  thirty  minutes.  Lives  for  twelve  to  seventeen  days  when 
dried.  Direct  sunlight  kills  it  in  thirty  minutes.  Its  thermal 
death -point  is  58°C.  in  thirty  minutes.  It  is  a  facultative  aerobe; 
grows  at  ordinary  temperature,  but  better  at  37°C. 

Cultures. — Grows  on  all  the  common  culture  media,  but  more 
slowly  than  the  colon  bacilli.  Gelatine  cultures  resemble  typhoid. 
The  growth  in  this  media  (which  it  does  not  liquefy)  produces  no 


DYSENTERY  BACILLUS  179 

pellicle,  but  a  sediment.  Indol  is  not  produced,  and  milk  is  first 
mildly  acid  and  then  faintly  alkaline,  though  not  coagulated.  On 
potato  it  grows  sparingly,  often  turning  it  brown.  The  Shiga 
type  ferments  glucose,  but  no  other  sugar.  The  Flexner  type 
ferments  glucose,  dextrine,  and  mannite,  but  not  lactose.  The 
latter  type  produces  more  acid  than  the  former,  and  both  are 
best  agglutinated  with  their  corresponding  serums. 

Habitat. — In  living  bodies  the  organism  is  found  solely  in 
mucous  discharges  from  the  bowels.  In  the  dead  it  is  found  in 
the  lymph-glands.  If  it  reaches  the  circulation,  it  appears  to  be 
rapidly  destroyed  by  the  blood.  It  has  been  discovered,  however, 
in  the  body  of  a  foetus  delivered  from  a  woman  with  the  disease. 
The  organism  must  have  passed  the  placenta  of  the  mother.  The 
disease  is  spread  by  water,  food  and  personal  contact  and  by 
carriers,  and  it  may  become  epidemic  in  large  institutions. 

Pathogenesis. — The  typical  lesions  caused  by  the  organism 
vary  from  a  mere  hyperaemia  to  a  superficial  necrosis  of  the  lym- 
phoid  structures,  which  may  be  extensive.  Peyer's  patches  are 
slightly  swollen  but  not  ulcerated.  The  descending  colon  and 
sigmoid  are  oftenest  attacked.  The  necrotic  masses  separate, 
leaving  shallow  ulcers.  The  lymph  structures  are  engorged  with 
polynuclear  leucocytes.  No  marked  lesion  is  found  in  the 
spleen.  The  liver  and  kidneys  often  undergo  marked  parenchy- 
matous  degeneration.  The  bacilli  being  possessed  of  a  powerful 
endotoxin,  so  that  dead  cultures,  if  injected  under  the  skin  cause 
marked  local  and  general  reactions.  Like  the  pyocyaneus  bacillus, 
this  organism  undergoes  auto-digestion  in  bouillon,  which  leaves 
the  latter  highly  toxic  owing  to  the  liberation  of  the  toxins. 
Laboratory  animals  quickly  succumb  to  injection  of  this  organism, 
injection  producing  a  marked  reaction  in  the  colon,  a  phenomenon 
suggesting  that  there  is  a  predilection  for  the  organ  and  that  the 
body  uses  it  as  an  excretory  organ  for  the  poison.  Dysentery 
cannot  be  induced  in  animals  by  feeding  cultures.  Poorly 
nourished  subjects  are  easily  infected  and  quickly  die.  Digestive 


l8o  BACTERIA 

disorders  favor  infection.  Death  may  be  due  to  toxaemiaor  ex 
haustion.  As  a  causal  agent  in  the  production  of  summer  diarr- 
hoeas of  children,  the  dysentery  bacillus  plays  a  part,  it  has 
been  isolated  from  the  stools  of  infants,  with  this  disease,  and 
their  sera  have  been  found  to  agglutinate  the  bacilli.  Never- 
theless it  is  known  that  other  bacteria  (streptococci,  etc.)  cause 
this  disease,  and  Weaver  found  that  "clinically  twenty-four  of 
our  ninety-seven  cases  of  ileocolitis  in  which  dysentery  bacilli 
were  discovered  did  not  differ  from  cases  in  which  dysentery 
bacilli  were  not  found. 

Immunity. — The  sera  from  convalescents  from  dysentery  show 
a  strong  bactericidal  action.  Anti-bodies  are  developed  by  in- 
fection and  by  artificial  inoculation  with  killed  cultures.  Kruse 
obtained  a  serum  from  horses  which  strongly  protected  a  guinea 
pig  against  a  lethal  injection  of  bacilli.  The  protective  property 
of  the  serum  is  due  to  its  bactericidal  action.  Here  the  ambo- 
ceptors  act,  but  only  in  the  presence  of  a  complement.  It  is 
possible  that  a  small  amount  of  anti-toxin  is  present  since  there  is 
some  reason  to  think  that  a  modicum  of  free  toxins  is  produced 
by  the  bacilli. 

Vaccination. — Shiga  tried  to  induce  (i)  passive  and  (2)  active 
immunity  in  many  individuals  by  injecting  both  anti-toxic  serum 
and  bacteria  into  them.  This  was  not  followed  by  a  lowered 
number  of  infections,  but  by  a  lowered  mortality.  A  serum  may 
be  produced  by  injecting  horses  with  several  dysentery  strains, 
called  a  polyvalent  anti-serum.  This  has  good  therapeutic  effects 
but  does  not  immunize  prophylactically. 

Agglutination. — The  serum  from  a  patient  suffering  from  either 
dysentery  or  summer  diarrhoea,  will,  after  about  a  week's  illness, 
agglutinate  bacilli.  This  property  is  not  always  present,  and 
its  absence  does  not  exclude  the  possibility  of  infection.  In 
performing  the  reaction,  both  Shiga's  and  Flexner's  type  of 
organism  should  be  used.  These  types  probably  bear  the  same 
relation  to  each  other  that  typhoid  and  paratyphoid  do. 


PYOCYANEUS  BACILLUS  l8l 

PYOCYANEUS  BACILLUS 

Bacterium  pyocyaneus. 

Bacillus  Pyocyaneus  (Fig.  50). 

Bacillus  of  Blue  Pus.     Also  called  Pseudomonas  pyocyanea. 

An  organism  of  minor  importance  as  a  pathogenic  agent,  that 
is  often  met  with  in  groin  or  axilla. 

Morphology  and  Stains. — Slender  rods,  often  growing  into 
thread-like  forms.  Exhibits  pleomorphism.  Sometimes  is 
rounded  and  cocci-like,  is  motile,  has  a  polar  flagellum,  and 


FIG.  50. — Bacillus  pyocyaneus.  ^(Kolle  and  Wassermann.) 

no  spores.  Stains  with  all  the  basic  aniline  dyes,  but  not  with 
Gram's  method. 

Oxygen  Requirements. — Usually  a  strict  aerobe. 

Cultures. — Grows  on  all  the  common  culture  media  luxuriantly, 
at  room  and  incubator  temperatures.  It  elaborates  two  pig- 
ments, a  water-soluble  greenish  bacteriofluorescein,  and  a  chloro- 
form soluble  pigment,  a  beautiful  blue  in  color,  called  pyocyanin. 
On  gelatine  plates  it  produces  yellowish-white  to  greenish,  yellow 
colonies  which  liquefy  the  gelatine,  causing  crater-like  excavations 
about  the  colonies.  Gelatine  stab  cultures  rapidly  liquefy  along 


l82  BACTERIA 

the  line  of  inoculation,  coloring  the  gelatine  greenish-blue,  and  a 
white  crumbly  deposit  forms  in  the  bottom  of  the  stab.  On  agar 
plates  it  produces  yellowish-white  colonies,  surrounded  by  a  zone 
of  bluish-green  fluorescence.  It  grows  luxuriantly.  In  agar 
tubes  it  multiplies  rapidly,  spreading  over  the  medium,  with 
wavy  thickened  edges.  The  agar  quickly  turns  a  dark  greenish- 
blue,  and  in  old  cultures  the  growth  changes  from  yellow  to 
greenish-blue. 

In  bouillon  it  is  very  dense  and  yellowish-green;  a  pellicle  forms 
on  the  surface,  and  a  sediment  is  deposited.  In  old  bouillon  cul- 
tures the  bacilli  undergo  autolysis  and  disappear.  In  milk  the 
growth  is  luxuriant,  the  casein  is  coagulated,  and  the  clot  is  ulti- 
mately digested.  The  reaction  is  alkaline.  On  potato  it  varies  in 
luxuriance,  often  being  slightly  elevated,  yellowish,turning  to  green. 
The  variance  in  growth  is  due  to  the  kind  of  potato  used.  Drying 
kills  the  organism  speedily;  four  hours  in  sunlight  also  destroys  it. 

Chemical  Activities. — No  gas  is  generated.  Besides  the  pig- 
ments (already  specified)  ammonia  is  produced,  also  a  peculiar 
enzyme  called  pyocyanase  by  Emmerich  and  Lowe,  which  not 
only  digests  gelatine  and  milk  curd,  but  its  own  and  other  bac- 
terial cells  as  well.  Old  cultures  are  poisonous;  a  haemolysin  is 
produced — an  endo-toxin,  and  a  soluble  toxin.  The  last-named 
toxin  stands  a  temperature  of  ioo°C.  Against  the  endo-toxin 
and  the  soluble  toxin  it  is  possible  to  prepare  an  anti-serum.  This 
may  protect  laboratory  animals. 

Pathogenesis. — Has  been  found  a  sole  cause  of  meningitis  and 
vegetative  endocarditis  in  man;  is  a  pyogenic  organism;  can  cause 
suppuration  anywhere  in  the  body;  produces  blue  pus;  is  patho- 
genic to  guinea  pigs;  and  its  virulence  can  be  raised  by  passing 
it  through  a  series  of  animals. 

Agglutination. — The  serum  of  infected  and  immunized  animals 
both  in  moderate  dilution  causes  agglutination  of  bacilli.  It  is 
possible  to  use  bacterins  of  this  germ.  Bactericidal  substances 
develop  by  the  use  of  killed  cultures. 


BACILLUS   OF   SOFT  CHANCRE  183 

BACILLUS  OF  SOFT  CHANCRE 

Bacterium  Ulceris  Chancrosi  (Ducrey). 

Streptobacillus  of  Soft  Chancre. 

Morphology  and  Stains. — A  small  thin  bacterium  .5;*  broad, 
1.5/1  long,  growing  in  chains  with  polar  staining,  which  can  be 
demonstrated  in  sections  of  chancroids  without  much  difficulty. 

This  organism  does  not  stain  by  Gram's  method,  but  by  Lofflers 
it  is  stained  with  ease. 

Cultures  are  hard  to  make.  It  grows  best  in  serum  agar,  and 
blood  agar  in  faint  colonies  that  are  not  very  characteristic.  In 
condensation  water  of  agar  it  grows  feebly. 

In  sections  and  in  pus  the  organism  is  frequently  found  in  the 
interior  of  leucocytes. 

By  aspirating  pus  from  buboes  and  planting  it  on  old  but  moist 
blood  agar  plates,  cultures  may  be  obtained. 

Pathogenesis. — From  an  old  culture  of  over  ten  generations 
typical  ulcerations  were  produced  in  man.  The  organism  is  feeble 
and  quickly  dies  in  culture  media  or  in  contact  with  mild 
antiseptics. 

ANTHRAX  BACILLUS 

Bacillus  Anthracis. 

Anthrax  Bacillus  of  Koch  (Fig.  51). 

Practically  the  first  pathogenic  organism  to  be  isolated.  This 
was  accomplished  by  Dr.  Robert  Koch.  It  is  the  cause  of  a  wide- 
spread malignant  disease,  variously  called  Anthrax,  Charbon,  or 
Splenic  Fever.  Animals  and  man  are  infected  by  it,  and  its  action 
is  often  rapidly  fatal. 

Morphology  and  Stains. — In  animal  tissues  this  organism  ap- 
pears as  a  large  rod  3-10^  long,  and  1-1.2;*  wide.  Is  of  ten  in  pairs 
or  chains.  In  fresh  specimens  the  ends  of  the  rods  are  rounded; 
when  older,  the  ends  become  square  or  concave.  Often  they  have 
faint  capsule  surrounding  them.  In  culture  media  they  exhibit 


1 84 


BACTERIA 


spores  and  grow  in  long  threads,  these  threads  form  long  spirally 
twisted  masses,  like  locks  of  wavy  hair.  No  flagella  are  formed, 
and  the  organism  is  not  motile.  In  old  cultures,  bizarre  involu- 
tion forms  are  found.  It  stains  well  with 
all  the  common  basic  dyes  and  by  Gram's 
method. 

Oxygen  Requirements. — Is  a  facultative 
anaerobe,  but  grows  much  better  in  the 
presence  of  oxygen.  If  oxygen  is  excluded, 
no  liquefaction  occurs. 

Temperature. — Grows  between  i4°C.  and 
45°C.;  best  at  37°C.     Spores  are  formed,  if 
oxygen  is  present,  between  i5°C.  and  4o°C. 
Sporulation  is  more  rapid  at  37°C.     Spores 
withstand  high  temperature  (dry)  for  a  long  time,  ioo°C.  for  one 
hour.     The  bacillus  itself  is  killed  at  7o°C.,  moist  heat,  in  one 


.  FIG.  51.— Anth- 
rax bacilli  in  blood. 
(Greene's  Medical 
Diagnosis.) 


FIG.  52. — Anthrax  bacilli  growing  in  a  chain  and  exhibiting  spores, 
and  Wassermann.) 


(Kolle 


minute.     The  thermal  death-point  may  be  put  down  for  the 
organism,  at  ioo°C.  steam,  for  five  minutes. 


ANTHRAX  BACILLUS  185 

Vital  Resistance. — Highly  resistant  to  chemicals,  light  and  dry- 
ing. Spores  resist  5  percent  carbolic  solution  for  days  (Esmarch), 
but  i-iooo  corrosive  sublimate  for  only  a  few  hours.  They  also 
resist  formaldehyde  and  sulphur  for  a  long  time,  and  withstand 
light.  A  2  percent  fresh  solution  of  H2O2  kills  spores  in  three 
hours.  Three  and  one-half  hours'  exposure  to  bright  sunlight 
killed  the  spores  if  oxygen  was  not  excluded  (Dieudonne)  (Fig.  52). 

Sporulation  Phenomena. — At  i2°C.  spores  are  formed  if  oxy- 
gen is  present.  The  most  favorable  temperature  for  sporulation 
is  that  of  the  body  (37°C.).  Spores  are  never  found  in  the  bodies 
of  living  or  dead  animals  if  they  remain  unopened,  and  oxygen 
is  excluded.  If  bacilli  are  cultivated  at  42°C.  for  a  long  time  and 
frequently  reinoculated,  on  fresh  media,  the  ability  to  form  spores 
is  lost  even  if  grown  again  at  3o°C.  (Phisalix).  If  cultivated 
upon  media  containing  carbolic  acid  and  hydrochloric  acid,  the 
ability  to  sporulate  may  be  lost. 

Chemical  Activities. — Acetic  acid  is  formed,  as  is  H^S.  Lique- 
fying, milk  coagulating,  and  milk  digesting  enzymes  are  formed. 
Toxins  have  not  been  isolated,  but  may  be  produced. 

Habitat. — Only  found  where  infected  animals,  hides,  and  hair 
have  been.  Fields,  hay,  bristles,  hides,  manure,  etc.,  have  been 
found  to  contain  bacilli.  Drinking  water  may  be  polluted  by  tan- 
neries and  the  bodies  of  dead  animals.  Meadows  and  fields  may 
be  contaminated  for  years.  From  the  buried  bodies  of  infected 
animals  anthrax  spores  may  be  brought  to  the  top  of  the  soil  by 
earth-worms. 

Cultures. — Grows  exceedingly  well  on  all  culture  media  in  the 
air.  On  gelatine  it  grows  in  whitish  round  colonies,  rapidly  sink- 
ing into  the  gelatine,  due  to  the  liquefaction.  The  liquid  medium 
is  turbid.  The  interior  of  the  colony  is  crumbly.  When  magni- 
fied, the  colonies  seem  to  be  made  up  of  tangled  waving  bundles, 
like  locks  of  hair,  especially  about  the  periphery.  In  gelatine  stab 
cultures  the  growth  is  luxuriant  and  rapid;  the  medium  is  liquefied 
more  rapidly  at  the  top,  and  finally  a  crater  is  formed;  before  this 


1 86  BACTERIA 

appears,  lateral  hair-like  outgrowths  are  seen  in  the  gelatine.  At 
the  bottom  of  the  crater  a  white  crumbly  mass  is  formed,  but  no 
pellicle.  On  agar  plates,  small  whitish  colonies  develop  which 
are  elevated  and  round.  When  magnified,  wavy  hair-like  growths 
appear  on  the  edge,  caused  by  many  twisted  parallel  chains  of 
bacilli  (Fig.  53). 

In  agar  stab,  the  growth  is  more  luxuriant  near  the  top;  lateral 
filamentous  branches  are  seen  along  the  stab  line.  In  agar 
streak  the  colonies  are  abundant,  thick  and  fatty;  have  tangled 
edges,  and  the  water  of  condensation  is  cloudy.  In  bouillon,  it 


FIG.  53. — Anthrax  bacilli.     Cover-glass  has  been  pressed  on  a  colony  and 
then  fixed  and  stained.     (Kolle  and  Wassermann.) 

forms  homogeneous  flocculi,  which  precipitate,  leaving  the  bouillon 
clear.  A  fragile  pellicle  is  formed.  In  milk,  it  multiplies  rapidly, 
the  proteids  are  coagulated,  generally  rendered  acid,  and  later  the 
coagulum  is  dissolved.  Potato  cultures  are  likewise  luxuriant. 
The  growth  is  elevated,  dull  in  lustre,  and  the  outline  is  wavy. 

Pathogenesis. — The  anthrax  bacillus  increases  so  rapidly,  and 
so  luxuriantly,  that  it  has  been  supposed  to  cause  death  merely 
by  mechanically  overwhelming  the  animal :  absorbing  nutriment 
and  oxygen,  and  blocking  capillaries.  Its  action  is  certainly  not 


ANTHRAX  BACILLUS  187 

purely  toxic,  as  it  causes,  not  a  toxaemia,  but  a  bacteriaemia.  It  is 
especially  virulent  for  man,  sheep,  cattle,  goats,  rabbits,  guinea 
pigs,  mules,  and  horses.  Rats  rarely  succumb.  Pigeons, 
chickens,  and  dogs  are  immune.  If  frogs  are  kept  at  a  tempera- 
ture of  3o°C.  they  become  susceptible  to  infection.  At  their 
normal  temperature  they  are  immune.  The  disease  produced  by 
this  organism  is  known  variously  in  different  countries  as  Anthrax, 
Splenic  fever,  Woolsorter's  disease,  Malignant  pustule,  and  Char- 
bon.  It  frequently  devastates  vast  herds  of  sheep,  cattle,  and 
goats,  and  is  often  a  pestilence  in  European  countries,  China,  and 
South  America.  It  appears  sporadically  in  the  United  States. 
Its  origin  in  this  country  can  usually  be  traced  to  infection  from 
hides  or  hair  imported  from  abroad.  The  disease  has  been 
contracted  from  using  shaving  brushes  made  of  insufficiently 
sterilized  bristles.  In  man  it  is  frequently  fatal.  The  infection  is 
first  manifest  as*  a  small  carbuncle  or  pustule,  from  this,  rapid 
general  infection,  as  a  rule,  ensues.  In  man  and  animals  anthrax 
bacilli  may  be  transmitted  from  mother  to  foetus  via  the  placenta. 
The  organism  is  found  in  enormous  numbers  in  infected  bodies, 
investing  all  the  organs  and  the  blood.  Pus  is  produced  by 
necrosis  of  tissue.  Infection  is  accompanied  by  a  high  leucocyto- 
sis  and  fever.  There  is  often  congestion  of  the  lungs;  also  an 
intense  friability  of  the  splenic  pulp,  and  all  the  glands  of  the  body 
become  enlarged,  and,  at  times,  many  of  them  suppurate.  In 
woolsorter's  disease,  the  bacilli  are  inhaled,  and  lung  lesions 
result. 

Immunity. — It  is  possible  to  immunize  animals  against  infection 
with  anthrax  by  means  of  vaccines.  By  this  means  the  lives  of 
many  thousands  of  domestic  animals  have  been  saved.  The  vac- 
cines are  made  by  growing  the  bacillus  at  42°C.  for  various  lengths 
of  time  to  attenuate  them.  An  anti-serum  has  been  produced  by 
repeated  injection  of  toxins  and  of  sporeless  rods.  It  is  used 
locally  around  a  pustule,  and  in  doses  of  50-100  c.c.  intravenously. 
It  is  anti-toxic  and  seems  to  stimulate  phagocytosis. 


I 88  BACTERIA 

TETANUS  BACILLUS 

Bacillus  Tetani. 

Tetanus  Bacillus  (Fig.  54). 
Lockjaw  Bacillus. 

First  seen  by  Nicolaier,  and  isolated  in  pure  culture  by  Kitasato. 

Morophology  and  Stains. — Rod-shaped.     Varying  from  i.2{j,  in 

length,  to  very  long  threads  of  20  to  40;*.     Sometimes  grow  in 


FIG.  54. — Tetanus  bacilli  showing  end  spores.     (Kolle  and  Wassermann.) 

chains;  frequently  appear  like  short  drum-sticks  with  a  spore  at  one 
end,  which  is  either  round  or  oval.  At  times,  the  bacilli  in  chains 
sporulate.  The  organism  is  motile;  possesses  numerous  flagella 
(from  50  to  100)  peritrichously  arranged;  stains  well  with  all  the 
common  basic  aniline  dyes,  and  retains  the  color  in  Gram's 
method  (Fig.  55). 

Oxygen  Requirements. — Strictly  anaerobic  when  freshly  iso- 
lated from  earth  or  wounds,  but,  after  long  cultivation  on  culture 
media,  it  becomes  more  tolerant  to  small  amounts  of  oxygen. 

Temperature. — Grows  best  at  37°C.    Below  i4°C.  not  at  all. 

Vital  Resistance. — Spores  resist  8o°C.  for  an  hour.     This  fact 


TETANUS  BACILLUS  189 

enabled  Kitasato  to  kill  all  other  organisms,  except  their  spores,  in 
pus.  Six  days'  exposure  to  direct  sunlight  is  needed  to  kill  the 
spores.  The  thermal  death-point  is  best  considered  as  ioo°C. 
for  one  hour.  They  are  killed  in  two  hours  by  5  percent  phenol 
+  .5  percent  HC1  and  in  thirty  minutes  by  i-iooo  HgCl2 
-f  .5  percent  HC1. 

Chemical  Activities. — Ferments  sugar;  produces  gas,  indol, 
alkali,  and  H^S.  which  gives  to  the  culture  an  odor  of  burnt  garlic 
or  onion;  marsh  gas,  CO2,  and  nitrogen  are  produced.  Gelatine  is 
liquefied.  The  most  important  product  of  growth  is  the  highly 
poisonous  complex  toxin,  which  is  made  up  of  tetanolysin,  and 
tetanospasmin;  the  latter  has  a  great  affinity  for  nerve  tissues. 
This  toxin  is  soluble  in  water,  and  can  be  separated  from  it  by 
means  of  ammonia  sulphate. 

Habitat. — Is  found  in  garden  soil,  hay,  manure,  and  dust. 
Has  been  found  in  cobwebs,  on  weapons,  in  cartridges,  and  in  the 
faeces  of  man  and  of  animals.  It  has  been  isolated  from  bronchi  in 
a  case  of  rheumatic  tetanus  in  which  there  was  no  lesion  in  the 
body  (Carbon  and  Perrors).  In  disease  it  is  found  in  the  infected 
wound,  generally  in  a  deeply  punctured  one,  which  is  usually  puru- 
lent and  contains  but  few  bacilli.  Puerperal  tetanus,  and  tetanus 
of  the  new-born,  are  but  varieties  of  the  disease,  dependent  upon 
the  site  of  infection  whether  of  the  placenta  or  umbilical  cord. 
Tetanus  sometimes  occurs  spontaneously,  without  a  sign  of  injury 
anywhere.  Sheep  and  goats  are  susceptible  to  infection,  so.  are 
guinea  pigs  and  rabbits.  Horses  are  peculiarly  susceptible.  Soil, 
or  manure,  getting  into  wounds,  is  often  a  cause  of  tetanus.  Cow- 
dung  poultices,  mud  dressings,  or  cobweb  applications  to  stop 
haemorrhages,  have  also  caused  the  disease.  Tetanus  following 
vaccination  may  be  due  to  infected  virus,  the  latter  becoming  in- 
fected from  the  faeces  of  the  vaccine-producing  cows  but  more 
commonly  is  due  to  dirt  getting  into  vaccination  wounds. 

Cultures. — This  organism  is  difficult  to  grow,  and  always 
requires  an  atmosphere  of  hydrogen. 


I 90  BACTERIA 

/ 

On  gelatine  plates,  the  colonies  appear  first  as  minute  white 
specks,  which  slowly  liquefy  the  medium.  As  it  grows,  hair-like 
threads  branch  out  into  the  medium,  and  the  colony  resembles 
the  periphery  of  a  chestnut  burr;  later,  the  white  appearance 
changes  to  yellow.  In  gelatine  stab  the  growth  is,  at  first, 
whitish  along  the  line  of  the  needle,  eventually  the  gelatine 
becomes  liquid,  and  a  bubble  of  gas,  partly  filled  with  whitish- 
cloudy  liquid  gelatine,  appears.  On  agar  plates  the  colonies  are 
ragged,  and  are  surrounded  by  delicate  out-spreading  filaments. 
In  deep  stab  culture,  down  in  the  agar  and  remote  from  the  top, 
a  spreading  tree-like  form  appears,  with  spike-like  growths  in  the 
agar.  Blood  serum  is  sometimes  liquefied.  Bouillon  is  uniformly 
clouded,  gas  is  generated  if  sugar  is  present,  and  toxin  is  produced. 
Milk  is,  generally,  not  coagulated. 

All  cultures  of  tetanus  must  be  grown  under  an  atmosphere  of 
hydrogen    in    media,    from   which   all   free 
oxygen  has  been  driven  by  boiling,  or  else 
abstracted  by  a  mixture  of  pyrogallic  acid 
and  sodium  hydrate.     It  is  possible  to  culti- 
vate the  organism  under  mica  covering,  or 
paraffine  poured  upon  freshly  boiled  media. 
If  sterile  glass  tubing  is  filled,  with  agar  or 
JTIG  rr  —Tetanus     gelatme>  and  inoculated  with  tetanus  bacilli, 
bacilli  showing  peri-     then  sealed,  colonies  will  develop,  as  perfect 

ril?u°.US  wgdla'     anaerobic    conditions    are    thus    obtained. 
(Kolle  and  Wasser- 

mann.)  Often   the  organism  grows  best  m  the  pres- 

ence of  saprophytic  ones.  Strongly  patho- 
genic organisms  do  not  grow  well  in  culture  media,  while  com- 
paratively non- virulent  ones  grow  very  well. 

Pathogenesis. — Tetanus  may  follow  any  wound,  no  matter  how 
insignificant,  though  deeply  punctured  ones,  caused  by  nails  or 
splinters,  are  more  often  followed  by  tetanus  infection,  especially 
if  the  puncture  is  sealed  by  blood  clots  or  pus,  and  so  creating  an 
anaerobic  condition  necessary  for  growth.  If  the  wound  is  on  the 


TETANUS  BACILLUS  IQI 

face  or  hand,  tetanus  symptoms  more  quickly  supervene,  while  if 
the  wound  is  on  the  foot,  these  are  apt  to  be  delayed.  The  sooner 
the  symptoms  appear  after  the  reception  of  the  injury,  the  more 
likely  will  the  disease  be  virulent  and  fatal.  If  spores  are  washed 
free  from  toxin,  according  to  Viallard  and  Rouget,  and  then  injected 
into  a  susceptible  animal,  they  do  not  cause  tetanus,  but  are  taken 
up  by  the  phagocytes.  In  other  words,  the  rods  not  the  spores 
produce  toxin.  Necrotic  tissue  in  wounds  favors  infection  with 
tetanus,  since  it  helps  to  fulfil  anaerobic  conditions,  and  in  some 
way  hinders  phagocytosis.  Aerobic  bacteria  favor  tetanus  infec- 
tion by  absorbing  the  free  oxygen  which  prevents  the  growth  of 
tetanus  organisms.  Free  oxygen  never  kills  the  organism  or  its 
spores,  but  merely  prevents  their  development.  Wounds  that 
have,  apparently,  healed,  may  be  the  origin  of  tetanus.  The  toxin 
is  produced  rapidly  in  wounds,  or  what  is  more  likely,  some  is 
introduced  with  the  bacilli  and  other  dirt.  Kitasato  found,  in  the 
case  of  mice,  that  if  bacilli  were  introduced  in  the  skin,  near  the 
tail,  and  in  an  hour  the  whole  area  was  excised,  and  the  wound 
cauterized,  fatal  tetanus  nevertheless  supervened.  Rheumatic 
tetanus  follows  pulmonary  infection. 

As  related  in  the  chapter  on  toxins,  the  mode  of  disease  produc- 
tion is  as  follows :  The  toxin  is  conveyed  from  the  wound  by 
means  of  the  motor  nerves  to  the  central  nervous  system  affect- 
ing the  motor  elements.  It  causes  microscopic  degeneration  of 
the  fibers  and  cells  of  the  motor  apparatus.  Death  is  caused 
either  by  a  spasm  of  the  glottis  or  diaphragm,  or  by  cardiac 
failure  and  exhaustion.  A  local  manifestation  merely  affecting 
certain  groups  of  muscles  may  occur.  Laking  of  the  blood  by 
tetanolysin  found  in  the  bodies  dead  from  tetanus  is  a  well- 
known  phenomenon.  In  fatal  cases,  toxin  may  be  demonstrated 
in  the  bladder  by  injecting  the  urine  into  mice,  causing  in  them 
tetanic  symptoms.  Various  groups  of  muscles  are  affected  in 
tetanic  seizures.  The  muscles  of  the  jaw,  if  affected,  cause 
trismus;  if  those  of  the  back  are  involved  the  individual  suffers 


I 92  BACTERIA 

from  opisthotonos.  The  seizures  may  be  constant  or  tonic;  or  con- 
vulsive and  violent,  then  they  are  designated  as  clonic. 

Immunity. — Metchnikoff  claims  that  the  only  natural  immu- 
nity possessed  by  man  against  tetanus  resides  in  his  leucocytic 
powers  of  defense.  Susceptibility  of  the  natural  receptors  of  the 
nerve  cells  for  the  toxin,  and  the  degree  of  affinity,  constitutes  the 
cause  of  intoxication,  its  degree,  and  ultimate  result.  Affinity  for 
the  receptors  of  other  less  vital  organs,  on  the  part  of  the  toxin, 
establishes  a  means  of  natural  defense.  Acquired  immunity  is 
dependent  upon  the  formation  of  anti-toxin.  The  anti-toxin, 
formed  by  susceptible  animals  injected  with  tetanus  toxin,  is 
chiefly  useful  and  valuable  as  a  prophylactic  measure.  An  epi- 
demic of  puerperal  tetanus  in  a  lying-in  hospital  was  checked  by 
its  use.  Sprinkling  dry  powdered  anti-toxic  serum  on  wounds  in- 
fected with  tetanus  bacilli,  or  toxin,  prevented  infection  (Calmette 
and  McFarland).  The  anti- toxin  may  be  injected  either  into  the 
substance  of  the  brain  in  cases  of  well  developed  tetanus,  or  into 
the  cerebrospinal  fluid,  in  the  hope  of  neutralizing  the  toxin  not 
already  in  firm  combination  with  the  nervous  elements.  Large 
nerves  near  the  infecting  wound  may  be  injected  with  anti-toxin 
in  the  hope  of  binding  the  toxin  already  in  combination  with  the 
nerve  cells  (see  page  74) . 

Female  mice  immunized  against  tetanus  toxin,  transmit  a  great 
amount  of  immunity  to  their  offspring.  The  milk  of  an  immun- 
ized mouse  also  causes  a  passive  immunity  in  other  young  that  are 
suckled  by  her. 

BACILLUS  OF  MALIGNANT  (EDEMA 

Bacillus  (Edematis  Maligni.  . 

Vibrion  septique. 
Bacillus  of  Malignant  (Edema. 

Morphology  and  Stains. — Thickish  rods,  resembling  tetanus 
and  symptomatic  anthrax  bacilli,  with  a  tendency  to  grow  in  long 


BACILLUS    OF    MALIGNANT    (EDEMA  193 

threads.  It  is  actively  motile,  and  is  possessed  of  numerous  peri- 
trichous  flagella.  Spores  are  found  which  may  be  either  equatori- 
ally  or  polarly  situated.  This  organism  is  readily  stained  by  the 
ordinary  methods,  but  not  by  Gram's. 

Chemical  Activities. — Milk  is  coagulated,  digested,  and  the 
reaction  is  amphoteric.  Abundant  alkali  is  formed  at  times; 
albumin  is  decomposed,  forming  fatty  acids,  leucin,  an  oil,  and  an 
offensive  odor.  CO2N.  and  marsh  gas,  are  also  formed. 

Habitat. — It  is  found  in  soil,  dust,  manure  and  dirty  water  and 
is  widely  distributed. 

Cultures. — This  organism  is  a  strict  anaerobic,  and  grows  well 
in  most  culture  media,  at  incubator  or  room  temperature.  On 
gelatine  plates  colonies  develop  on  the  surface  (under  hydrogen) 
in  tiny  shining  white  bodies,  which  upon  magnification  are  found 
to  be  filled  with  a  grayish-white  substance  composed  of  melted 
gelatine,  and  long  tangled  filaments.  The  edges  of  the  colonies 
are  fringed.  In  gelatine  stab  cultures  (made  in  liquid  gelatine, 
which,  after  inoculation,  is  rapidly  solidified  in  ice  water)  a 
globular  area  of  liquefaction  occurred.  If  sugar  is  added,  active 
fermentation  takes  place,  with  the  production  of  large  amounts  of 
offensive  gas.  It  grows  well  on  agar,  in  bouillon,  and  in  milk. 

Pathogenesis. — Is  pathogenic  for  man,  horses,  sheep,  dogs,  rab- 
bits, calves,  pigs,  goats,  rats,  mice,  and  guinea  pigs.  Cattle  are 
said  to  be  immune.  When  bacilli  are  applied  to  a  scratched  sur- 
face, infection  is  not  likely  to  occur,  as  free  oxygen  seems  to  inhibit 
the  growth;  if,  however,  the  wound  is  deep,  rapid  infection  follows, 
young  domestic,  and  laboratory  animals  dying  within  forty-eight 
hours.  The  bacillus  produces  a  moderate  quantity  of  toxin  and 
has  an  antagonistic  action  on  leucocytes.  In  man,  the  clinical 
manifestation  of  infection  with  this  organism  is  known  as  malig- 
nant oedema.  Infection  has  followed  penetrating  wounds  of  the 
body,  by  dirty  tools,  nails,  splinters,  bullets,  etc.  The  disease 
is  often  quickly  fatal.  It  produces,  frequently,  rapid  moist 
gangrene. 

13 


194 


BACTERIA 


The  organisms  and  spores  are  not  so  very  resistant  as  any 
strong  germicide  or  a  temperature  of  100°  acting  a  few  minutes 
will  kill  them. 

SYMPTOMATIC  ANTHRAX  BACILLUS 

Bacillus  Chauvoei. 

Bacillus  of  Symptomatic  Anthrax. 
Rauschbrand  Bacillus  (Figs.  56  and  57). 


FIG.  56.— Rauschbrand  bacilli  showing  spores.     (Kolle  and  Wassermann.) 


The  cause  of  symptomatic  anthrax,  black-leg,  or  quarter-evil, 
in  cattle. 

Morphology  and  Stains. — This  is  a  large  organism,  .5^  in  width, 
and  3  to  5/x  in  length.  It  has  rounded  ends,  and  grows  in  pairs, 
but  not  in  strings  or  chains.  It  is  motile,  and  has  many  peritri- 
chous  flagella.  When  stained  for  spores,  these  bodies  may  be 
found  distending  the  organism  in  the  middle  or  at  the  end,  and  the 
bacillus  assumes  a  drum-stick,  or  spindle  shape.  Often  chromo- 
philic  granules  are  present;  involution  forms  also  appear,  and  are 
of  enormous  size.  This  organism  stains  with  all  the  common 


SYMPTOMATIC   ANTHRAX  BACILLUS  1 95 

stains,  but  not  by  Gram's  method.  They  may  be  seen  in  an 
unstained  condition  in  blood  or  other  fluids. 

Habitat. — This  bacillus  is  found  not  only  in  the  diseased  tissues 
and  dead  bodies  of  infected  animals,  but  also  in  infected  pas- 
tures, soil,  hay,  etc. 

Temperature  Requirements. — It  is  best  cultivated  at  body 
temperature,  but  grows  anywhere  between  i8°C.  and  37°C.  The 
spores  resist  boiling  for  half  an  hour  but  the  vegetative  rod  is 
killed  by  ioo°C. 


FIG.  57.— Rauschbrand  bacillus  showing  flagella.     (Kolle  and  Wassermann.) 

Cultures. — It  is,  like  tetanus  and  malignant  oedema  organisms, 
a  strict  anaerobe.  On  gelatine  it  grows  in  roundish  whitish  colo- 
nies in  a  delicate  tangled  mass,  with  projecting  filaments.  The 
gelatine  is  liquefied,  and  bubbles  of  gas  are  formed  in  stab  cultures. 
A  sour  odor  is  emitted  from  cultures;  i  to  2  percent  of  sugar  is 
required  for  successful  cultivation;  or  5  percent  of  glycerine  will 
answer.  On  agar  the  growth  is  marked;  gas  is  produced,  and 
acidous  odors  evolved.  In  bouillon  it  grows  rapidly.  Large 
masses  of  the  organism  sink  to  the  bottom,  gas  is  formed,  and  the 
medium  is  clouded.  Milk  affords  a  good  medium  for  the  growth 
of  the  organism,  but  the  casein  is  not  coagulated. 


ig6  BACTERIA 

Pathogenesis. — Young  cattle,  six  months  to  four  years  old, 
sheep,  goats,  rats,  mice,  and  more  especially  guinea  pigs,  are  sus- 
ceptible to  it.  Swine  are  immune,  while  dogs,  cats,  birds,  and 
rabbits  are  not  susceptible.  Man  is  immune.  It  causes  in  ani- 
mals peculiar  groups  of  emphysematous  crepitating  pustules,  fol- 
lowed by  emaciation  and  death.  These  areas  contain  dark  fluid, 
probably  broken-down  blood.  In  guinea  pigs  inoculation  is  fol- 
lowed by  death  within  thirty-six  hours.  The  site  of  inoculation 
is  found  to  be  cedematous,  and  contains  bloody  fluid.  The  bacilli 
are  mostly  found  at  the  site  of  the  inoculation,  but  later  in  the 
blood  in  every  part  of  the  body.  The  virulence  of  this  organism 
in  culture  media  is  soon  lost.  The  addition  of  lactic  acid  to  the 
cultures  increases  their  virulence. 

Immunity. — It  is  possible  to  decrease  the  virulence  of  this 
organism,  and  to  use  the  weakened  bacteria  as  a  vaccine  against 
infection.  To  attenuate  this  bacillus,  prolonged  exposure  to  heat, 
or  to  heat  and  drying  together  is  necessary.  Inoculation  with 
bacilli  treated  in  this  way  is  followed  by  a  mild  local  reaction, 
which  affords  complete  immunity  against  infection  with  virulent 
bacilli.  It  has  been  found  by  Kitt  that  the  muscles  of  an  infected 
animal,  if  subjected  to  a  high  temperature — 85°C.  to  9ocC. — 
afforded  complete  protection  to  the  animal  inoculated  with  them. 
It  is  best  to  use  a  weaker  vaccine  muscle  that  has  been  heated  to 
ioo°C.  for  two  hours,  in  order  to  protect  against  the  active 
vaccine.  Before  heating,  the  meat  is  ground.  When  used  as  an 
injection,  it  is  crushed  and  mixed  in  a  mortar  with  sterile  water. 
Guillod  and  Simon  found  that  this  means  of  preventative  inocula- 
tion reduced  the  death  rate  in  unprotected  animals  from  20  per- 
cent to  5  percent.  If  this  bacillus,  and  the  prodigiosus  bacillus 
are  injected  into  naturally  immune  animals,  death  will  often 
result. 

There  is  a  soluble  toxin,  anti-toxin  against  which  appears  in  ' 
immunized  animals.     The  toxin  may  be  used  for  prophylaxis. 
One  attack  confers  immunity. 


„  MEAT   POISONING  BACILLUS  IQ7 

MEAT  POISONING  BACILLUS 

Bacillus  Botulinus.     Van  Ermengen. 

Bacillus  of  Meat  Poisoning,  or  Botulism  (Fig.  58). 

Morphology  and  Stains. — This  bacillus  resembles  thick  vigor- 
ous rods,  4-9/4  long,  and  .9^  thick,  is  motile,  has  polar  spores,  and 
from  four  to  nine  peritrichous  flagella.  It  is  strangely  called  a 
saprophyte,  because  it  is  incapable  of  growth  in  the  body,  yet  its 
toxin  is  highly  poisonous  to  man  and  other  animals.  It  is  stained 
by  all  the  usual  basic  aniline  dyes,  and  by  Gram's  method. 


FIG.  58. — Bacillus  of  botulism.     (Kolle  and  Wassermann.) 

Habitat. — It  seems  probable  that  this  organism  occurs  in  the 
feces  of  animals,  especially  pigs,  from  which  source  it  can  gain 
access  to  the  ground,  to  vegetables,  or  to  the  meat  of  the  animal 
from  which  hams  are  cured  or  sausages  made.  While  originally  a 
disease  described  as  originating  from  improperly  cured  hams, 
botulism  has  been  known  to  follow  the  eating  of  tomatoes,  beans 
and  olives. 

Vital  Characteristics. — Is  an  anaerobe.  Its  thermal  death- 
point,  for  a  spore-bearing  organism,  is  low,  8o°C.,  for  an  hour. 


IQ8  BACTERIA 

Grows  only  in  media  that  are  alkaline,  and  is  capable  of  growth 
at  from  i8°C.  to  35°C.,  though  best  below  25°C.;  10  percent 
of  chloride  of  soda  checks  growth. 

Chemical  Activities. — It  can  produce,  at  room  temperature, 
a  water-soluble  toxin  sufficiently  stable  to  withstand  drying  of 
meat  if  not  exposed  to  sunlight,  and  not  destroyed  by  the  gastric 
juice.  It  is  destroyed  by  thorough  cooking  of  meat.  Milk  is 
not  coagulated,  grape  sugar  is  fermented,  and  a  foul,  sour  odor 
is  produced  in  a  culture.  It  liquefies  gelatine.  There  are  two 
varieties  of  the  germ,  A  and  B,  differing  in  the  quality  of  the 
toxin  produced,  both  having  the  same  physical  and  pathogenic 
properties  but  developing  different  anti-toxins. 

Cultures. — On  gelatine  plate,  that  contains  sugar,  colonies  are 
produced  that  are  coarse  and  prickly  in  appearance.  The  lique- 
faction of  the  gelatine  is  slow.  Bouillon  is  rendered  turbid.  The 
cultures  resemble  tetanus  and  malignant  cedema. 

Pathogenesis. — Its  pathogenic  action  is  marked,  but  only  by 
its  toxin,  which  has  a  decided  affinity  for  nervous  tissue.  The 
toxin  is  absorbed  from  the  intestinal  tract  unchanged  by  the 
gastric  juice.  In  this  it  differs  from  the  toxin  of  diphtheria  and 
tetanus.  If  the  toxin  is  mixed  with  the  emulsified  nerve  tissue, 
it  becomes  neutralized.  In  fatal  cases  of  infection,  the  gan- 
glionic  nerve  cells  are  degenerated.  Man  is  very  susceptible, 
while  cats  and  dogs  are  more  or  less  non-susceptible.  If  bacilli 
are  inoculated  into  animals,  they  do  not  proliferate.  A  men- 
ingitic  disease  of  horses  and  limberneck  of  fowl  are  believed  to 
be  due  to  this  intoxication.  Animals  that  recover  are  found 
to  have  developed  strong  anti-toxin  in  the  blood  serum. 

Immunity. — An  artificially  prepared  anti-toxin  has  been  found 
to  be  active^  and  is  of  use  in  treating  cases  of  poisoning  with  meat. 
The  correctly  typed  anti-toxin  should  be  used  or  that  made  by 
artificial  immunization  with  both  varieties  of  bacilli.  The  latter 
is  now  preferred  since  no  rapid  distinguishing  clinical  test  of 
types  is  available. 


GASEOUS   CEDEMA  BACILLUS 

GASEOUS  (EDEMA  BACILLUS 


199 


Bacillus  Capsulatus  Aerogenes.— Welch. 

This  description  covers  those  organisms  sometimes  described 
under  the  title  Bac.  perfringens  and  Bac.  enteriditis  sporogenes; 
they  are  in  all  probably  but  variants  from  this  type.  Other 
types  with  slight  variations  in  chemical  action  and  toxin  produc- 


FIG.  59. — B.  Aerogenes  capsulatus  of  Welch,  in  smear.     (Williams.) 

tion  were  noted  during  the  war.     It  is  this  group  which  was 
responsible  for  gas  infection  during  the  great  European  War. 

Morphology  and  Stains. — A  vigorous  plump  bacillus  3  to  47*  in 
length,  resembling  the  anthrax  bacillus,  and  is  usually  straight. 
It  forms  spores,  is  non-motile,  and  flagella  have  not  been  found. 
It  occurs  in  pairs,  and  in  chains.  In  old  cultures  involution  forms 
are  seen.  Spores  are  generally  equatorially  situated.  Is  colored 


200 


BACTERIA 


FIG.    60.— B, 
sulatus,    agar 
gas  formation. 


aerogenes  cap- 
culture   showing 
(Williams.) 


by  all  the  basic  dyes,  and  holds  the 
stain  in  Gram's  method.  Staining 
shows  that  it  possesses  a  capsule. 

Habitat. — The  soil,  the  intestines, 
and,  sometimes,  the  skin  of  man. 

Vital  Characteristics.— Vital  re- 
sistance is  low,  the  thermal  death- 
point  being  58°C.  with  ten  minutes' 
exposure,  while  spores  require  ten 
minutes'  boiling  for  killing.  It 
grows  best  at  body  temperature. 
Has  lived  for  one  hundred  days  on 
culture  media  in  the  incubator.  It 
is  an  anaerobe. 

Chemical  Activities. — Produces 
gas;  does  not  usually  liquefy  gela- 
tine, but  curdles  milk  (Fig.  60). 

Cultures. — Grows  best  in  neutral 
or  alkaline  media,  producing  abund- 
ant gas.  Colonies  appear  grayish 
or  brownish-white,  and  are  often 
surrounded  by  projections  which 
are  feathery  or  hair-like.  On  agar 
strict  anaerobic  conditions  are 
necessary  for  growth,  gas  bubbles 
appear  in  the  media,  and  the  agar 
may  be  forced  out  of  the  tube  in 
stab  cultures.  In  bouillon  it  grows 
under  anaerobic  conditions.  The 
growth  is  rapid,  bouillon  is  clouded, 
and  a  froth  appears  on  the  surface. 
After  a  few  days  the  medium  be- 
comes clear,  owing  to  the  sedi- 
mentation of  the  bacilli.  Growth 


GASEOUS    (EDEMA  BACILLUS  2OI 

occurs  best  in  sugar  bouillon,  which  becomes  strongly  acid.  In 
milk  the  growth  is  rapid  and  luxuriant;  the  proteids  are  coagu- 
lated. Anaerobic  conditions  must  be  observed.  On  potato  it 
grows  well,  producing  bubbles  in  the  water  which  may  cover 
the  potato  in  the  tube.  The  growth  appears  thin,  moist,  and 
grayish-white. 

Pathogenesis. — The  pathogenic  properties  of  this  organism  are 
limited.  It  is  not  able  to  endure  the  oxygen  of  the  circulating 
blood.  Grows  best  in  old  clots,  and  in  the  uterus.  It  produces 
gas  rapidly  in  some  cases  of  abortion  and  in  peritonitis  in  man, 
which  is  quickly  followed  by  death.  It  causes  gaseous  phleg- 
mons in  guinea  pigs,  and  injection  are  usually  fatal  to  birds. 
In  man  infection  has  followed  wounds,  and  delivery  of  the  child 
in  puerperal  cases.  It  produces  in  fatal  cases  the  condition 
known  as  frothy  organs — "  Schaumorgane. "  It  may  be  isolated 
from  infected  matter,  faeces,  etc.,  by  injecting  the  latter  into  a 
rabbit's  vein  and  then  killing  the  animal.  The  carcass  is  then 
placed  in  an  incubator  and  an  enormous  growth  of  the  organisms 
follows;  anaerobic  conditions  favorable  to  growth  are  obtained 
in  the  body  so  that  gas  distention  of  the  tissues  results;  from  the 
latter  pure  cultures  are  easily  obtained. 

Vincents  Angina  is  due  to  an  anaerobic  organism  of  two  stages, 
as  Bacillus  fusiformis  and  Spirochata  wncenti.  The  bacillus  is  a 
fusiform  irregularly  staining  pointed  rod,  3-12/4  long  by  .3-.8/* 
wide.  Under  cultivation  it  grows  out  into  forms  such  as  are  seen 
with  it  in  smears  from  the  diseased  throat,  that  is,  long,  wavy, 
uniformly  stained,  flexible,  pointed  ended  spirals.  The  bacillus 
forms  endospores  chiefly  at  the  end.  Obligate  anaerobe,  requir- 
ing serum,  ascitic  fluid  or  glycerine.  Colonies  delicate  and 
whitish.  Gas  in  glucose  media.  Litmus  milk  only  decolorized. 
Gives  a  foetid  odor  on  all  cultures.  No  specific  immunity  reac- 
tions known. 

These  same  spirals  have  been  found  in  abundance  in  many 
cases  of  ulcerative  stomatitis,  notably  the  variety  which  became 


202  BACTERIA 

well  known  during  the  late  war.  Their  connection  with  this 
so-called  "Trench  mouth"  is  not  so  generally  accepted  as  is  the 
case  with  ulcerative  angina  but  their  discovery  should  suggest 
remedies,  salvarsan,  silver  nitrate,  which  have  been  useful  in 
Vincents  Angina. 

SPIRILLACE^E 
CHOLERA  BACILLUS 

Vibrio  Cholerse.    Koch. 
Spirillum  Cholera  (Fig.  61). 
Cholera  Bacillus. 
Comma  Bacillus. 

Morphology  and  Stains. — Curved  or  bent  rods,  the  ends  not 
lying  in  the  same  plane.     This  bending  varies  greatly.     Under 


FIG.  61. — Cholera  spirilla.     (Kolle  and  Wassermann.) 

certain  conditions  of  growth  such  as  the  presence  of  alcohol,  or 
insufficient  albumin  or  oxygen  in  culture  media,  long  spiral  chains 
are  formed.  It  is  motile,  has  one  terminal  flagellum,  and  like 
other  members  of  this  family,  has  no  spores.  It  stains  well  with 
the  common  dyes  but  not  by  Gram's  method.  Dilute  fuchsin 


CHOLERA  BACILLUS  203 

stains  it  best.  Occasionally  involution  forms  are  developed, 
which  do  not  stain  well.  So-called  arthrospores  are  formed,  ac- 
cording to  Hlippe. 

Habitat. — It  is  said  to  exist  constantly  in  the  waters  of  the 
Ganges  in  India.  Is  found  in  contaminated  drinking  water, 
from  rivers,  lakes,  and  wells;  also  in  human  faeces,  which,  used  as 
manure,  infests  vegetables,  and  spreads  the  disease.  It  is  found 
in  the  intestines  during  cholera,  and  after  death  in  other  viscera. 
It  can  be  disseminated  by  convalescent  or  healthy  carriers; 
chronic  carriers  are  not  known. 

Vital  Resistance. — Is  extremely  sensitive  to  various  deleterious 
agencies.  Minute  quantities  of  mineral  acids,  and  other  chemical 
disinfectants,  as  well  as  light,  heat,  and  drying,  quickly  kill  it; 
i  percent  carbolic  kills  rapidly.  A  1-2,000,000  solution  of  cor- 
rosive sublimate  destroys  in  from  five  to  ten  minutes.  Its  thermal 
death-point  is  6o°C.  for  ten  minutes  (moist  heat). 

Chemical  Activities. — It  creates  indol  in  large  quantities,  and 
may  be  detected  in  peptone  cultures  merely  by  the  addition  of 
sulphuric  acid.  Laevorotatory  lactic  acid  is  produced  from  all  the 
sugars.  Gases  are  not  formed.  Yields  alkali  in  culture;  causes 
slight  coloration  of  potato,  and  produces  a  disagreeable  odor  in 
bouillon;  also  yields  H2S,  and  ferments  that  liquefy  gelatine. 
Bacteriolysins  and  invertin  are  also  produced,  as  well  as  a  toxin 
which  is  soluble  in  water.  The  most  powerful  toxin,  by  far,  is 
contained  in  the  cells  of  the  vibrio  themselves.  This  causes  death 
after  intraperitoneal  injection  in  guinea  pigs. 

Oxygen  Requirements. — It  is  a  facultative  aerobe;  its  growth, 
however,  without  oxygen  is  slow,  while  powerful  toxins  are 
formed. 

Temperature. — Grows  best  at  37°C.,  but  very  well  at  23°C. 
Does  not  grow  below  8°C. 

Cultures. — On  gelatine  plates  the  growth  is  characteristic. 
Small  yellowish- white  colonies,  which  rapidly  liquefy  the  gelatine, 
appear  in  twenty-four  hours.  As  the  colony  increases  in  size  it 


204  BACTERIA 

becomes  more  and  more  granular,  and  finally  the  whole  medium 
is  liquefied.  In  gelatine  tube  stab  culture,  the  growth,  at  first,  is 
not  characteristic;  but,  after  a  few  hours,  a  semi-spherical  depres- 
sion appears,  which  extends  downward,  and  resembles  a  large 
bubble  of  gas.  As  liquefaction  progresses,  the  whole  line  of  punc- 
ture disappears,  and  the  excavation  looks  cylindrical.  This  area 
becomes  cloudy.  On  agar  plates  the  colonies  are  elevated,  round 
and  white,  with  moist  lustre.  Deep  colonies  are  whetstone  shape. 
Old  agar  colonies  become  yellowish-brown.  Coagulated  blood 
serum  is  rapidly  liquefied  at  37°C.  Milk,  at  times,  is  coagulated. 
No  curdling  ferment  is  formed;  the  acid  produced  is  thought  to  be 
sufficient.  On  potato  the  growth  is  slow,  or  not  at  all,  if  the 
medium  is  acid.  If  the  potato  is  rendered  alkaline,  growth  occurs, 
with  a  moist  lustre,  slightly  elevated;  white  at  first,  later  becoming 
brown.  On  acid  fruits  it  will  not  grow.  In  bouillon,  after  sixteen 
hours,  a  diffuse  cloudiness  occurs,  with  the  formation  of  a  stiff 
pellicle,  which  in  some  cultures  becomes  wrinkled.  In  peptone, 
abundant  growth  takes  place,  with  the  production  of  indol  and 
nitrites.  If  a  few  drops  of  H2SO4  are  added,  a  beautiful  red 
appears  if  nitrites  are  present.  This  is  the  "  cholera  red  "  reaction. 
If  the  color  does  not  at  once  appear,  nitrites  must  be  added.  On 
blood  media  certain  strains  produce  distinct  hemolysis  (El  Tor). 
Pathogenesis. — Cholera  spirilla  are  pathogenic  for  man,  but 
only  under  experimental  conditions,  for  lower  animals  for  which 
guinea  pigs  may  be  taken  as  an  example.  If  the  stomach  of  the 
latter  is  rendered  alkaline  with  bicarbonate  of  soda,  and  a  bouillon 
culture  introduced,  choleraic  symptoms  will  follow  and  the  animal 
will  die.  If  cholera  spirilla  are  injected  into  the  peritoneum,  the 
animal  will  quickly  succumb  to  a  general  cholera  peritonitis. 
Young  rabbits  are  equally  susceptible.  When  cholera  spirilla  in 
culture  have  been  swallowed  by  man  (laboratory  workers),  either 
by  design  or  accident,  the  disease  has  followed,  sometimes  with 
fatal  results.  The  toxin  of  this  organism  is  intracellular  (an 
endo-toxin).  Old  cultures  become  pathogenic  through  a  bac- 


CHOLERA  BACILLUS  2O$ 

teriolytic  action,  by  which  the  cells  are  dissolved,  and  the  toxin 
liberated.  Filtrates  from  young  cultures  are  non-toxic.  If 
bouillon  cultures  are  killed  by  chloroform,  and  then  injected  into 
animals,  toxic  action  follows.  In  cholera  the  pathogenic  process 
is  mostly  confined  to  the  intestines.  Toxic  absorptions,  due  to  the 
liberation  of  toxic  products  by  the  bacteriolytic  action  of  serum, 
follow  later.  There  is  a  desquamation  of  the  epithelium  of  the 
bowel,  and  epithelial  flakes  found  in  the  watery  discharges  resem- 
ble rice  grains.  Peyer's  patches  may  become  slightly  swollen 
and  reddened,  and  later,  there  may  be  diphtheritic  necrosis  above 
the  iliocecal  valve,  and  often  a  parenchymatous  nephritis.  The 
vibrios  do  not  enter  the  blood. 

Diagnosis. — Bacteriological  diagnosis  of  cholera  is  accomplished 
by  examining  the  alvine  discharges.  A  mucous  flake  is  mixed 
with  some  peptone  solution,  this  is  incubated,  and  the  spirilla, 
if  present,  rapidly  grow  on  the  surface;  after  a  few  hours,  plates 
are  poured  from  this  surface  growth,  and  from  the  plates  liquefy- 
ing colonies  are  picked  out,  and  bouillon  cultures  made.  These 
are  tested  by  serum,  from  horses  artificially  immunized  by  inject- 
ing cholera  spirilla  into  them.  If  the  organism  under  examination 
(after  serum  mixed  with  2,000  to  3,000  parts  of  water  is  added) 
agglutinates,  it  is  considered  to  be  the  cholera  spirillum.  Both 
in  early  and  fatal  cases,  the  agglutinating  reaction  is  not  available, 
since  it  takes  some  time  for  the  agglutinins  to  form  in  the  blood. 
Under  the  chapter  on  immunity  an  account  of  the  PfeifTer  reaction 
is  given,  also  one  on  vaccination  against  cholera  infection,  by 
means  of  killed  cultures,  under  the  chapter  on  vaccines. 

Vibrios  Allied  to  the  Cholera  Vibrio 

Several  other  vibrios  have  been  discovered  that  resemble  the 
cholera  vibrio.  These  are  mostly  found  in  potable  water,  and 
though  in  many  respects  identical  with  the  cholera  vibrio,  they 
differ  in  essential  points,  i.e.,  pathogenicity,  and  in  their  agglutina- 
bility  with  specific  sera.  The  most  important  of  these  organisms 


206  BACTERIA 

are:  Vibrio  Metchnikovii;  Vibrio  proteus;  Vibrio  tyrogenum;  and 
Vibrio  schuylkilliensis.  There  are  no  important  pathogenic  mem- 
bers of  this  group  except  the  cholera  vibrio. 

GLANDERS  BACILLUS 

Bacterium  Mallei. 

Bacillus  Mallei. 

Glanders  Bacillus. 

Morphology  and  Stains. — Slender  rods  2  to  3/x.  in  length  con- 
taining no  true  spores,  but  shining  chromatophilic  bodies  (Babes- 
Ernst  granules).  In  old  culture,  long  club-like  threads  appear, 
which  exhibit  true  branching.  This  organism  is  not  motile,  and 
has  no  flagella.  It  is  stained  with  difficulty  by  ordinary  methods, 
and  not  at  all  by  Gram's  method. 

Vital  Activities. — It  is  a  facultative  aerobe,  growing  feebly  in 
the  absence  of  air,  and  best  at  37°C.,  in  glycerine  agar.  Resists 
drying  but  feebly.  Its  thermal  death-point  is  5S°C.,  ten  minutes' 
exposure. 

Chemical  Activities. — Produces  a  brown  pigment  on  potato, 
also  mallein,  and  a  little  indol  in  old  bouillon  cultures.  It  forms 
no  gas. 

Cultures. — On  gelatine  it  produces  small  punctiform  colonies 
that  are  white,  and  become,  after  a  time,  surrounded  by  a  distinct 
halo.  The  colonies  are  often  very  delicate  and  ragged.  The  gela- 
tine is  not  liquefied.  On  agar  the  growth  is  best  if  glycerine  is 
present,  but  is  not  characteristic.  Bouillon  cultures  cause  an 
abundant  sediment,  above  which  the  medium  is  clear.  Milk  is 
coagulated.  On  potato  the  growth  is  characteristic.  The  color 
is,  at  first,  yellowish-white  like  honey,  becoming,  finally,  reddish- 
brown.  The  potato  is  much  darkened. 

Pathogenesis. — This  organism  is  pathogenic  for  horses  and 
man;  50  percent  of  men  succumb  after  infection.  Horses,  asses, 
cats,  dogs,  sheep,  and  goats  are  susceptible  in  the  order  mentioned. 
Cattle  and  birds  are  immune.  In  horses  the  disease  is  known  as 


DIPHTHERIA  BACILLUS  207 

glanders,  or  farcy,  and  the  avenue  of  infection  determines  the 
clinical  form  of  the  disease.  The  mucous  membrane  and  the  skin 
are  the  chief  places  of  infection.  A  primary  ulcer  is  formed  in  the 
mucous  membrane  of  the  nose,  or  in  the  skin.  Subsequently,  the 
lymph-glands  and  the  lungs  may  be  infected.  Guinea  pigs  are 
easily  infected.  White  and  gray  mice,  and  rats  are  immune.  For 
purposes  of  diagnosis  guinea  pigs  are  inoculated,  but  care  must  be 
used,  as  several  fatal  cases  have  occurred  in  laboratory  workers, 
it  being  a  treacherous  organism  with  which  to  work.  In  infected 
animals,  it  produces  a  rapid  and  marked  inflammatory  reaction, 
with  the  formation  of  pus.  Certain  "buds,"  or  nodules  are 
formed,  which  are  between  an  abscess  and  a  tubercle  in  structure. 

The  diagnosis  of  doubtful  cases  may  be  made  by  injecting  the 
material  into  the  peritoneum  of  male  guinea  pigs.  A  violent 
suppurative  orchitis  occurs  from  which  the  rods  can  be  cultivated. 
The  poisons  are  endo-toxic. 

Agglutinations. — It  has  been  shown  by  McFadyean  that  the 
blood  of  infected  horses  exhibits  markedly  agglutinative  properties 
toward  the  glanders  bacilli.  Normal  horse  serum  clumps  often  as 
high  as  1-400.  Diagnostic  reactions  should  be  i-iooo,  supported 
by  complement  fixation  and  the  Mallein  test.  A  slight  immunity 
is  present  after  an  attack.  Complement  fixation  may  be  attained 
with  the  serum  of  an  infected  horse  by  using  an  antigen  of  glanders 
bacilli  grown  on  glycerine  broth,  killed  and  filtered. 

Mallein. — In  old  cultures  a  peculiar  tuberculin-like  substance 
(mallein)  is  formed  from  the  bodies  of  the  bacilli  themselves,  and 
in  the  bouillon.  This  is  thermostabile  and  if  injected  into  animals 
having  glanders,  produces  a  marked  reaction  (see  page  86). 

DIPHTHERIA  BACILLUS 

Corynebacterium  Diphtherias  (Loftier). 

Bacillus  Diphtheria. 
Klebs-Loffler  Bacillus. 
Diphtheria  Bacillus. 


208 


BACTERIA 


Morphology  and  Stains. — Long,  bent,  or  curved  bacilli  of 
irregular  contour,  frequently  clubbed  or  filiform  at  one  or  both 
ends ;  which  contain  chroma tophilic  granules,  and  often  exhibit  true 
branching ;  have  no  spores  or  flagella,  and  are  not  motile.  Accord- 
ing to  Wesbrook,  stained  bacilli  are  of  three  types;  (i)  granular 
(containing  the  Babes-Ernst  granules);  (2)  barred  like  a  striped 
stocking;  or  (3)  solid,  staining  uniformly  throughout.  The  pleo- 
morphic  differences  of  various  bacilli  are  most  characteristic,  and 
of  diagnostic  importance.  This  organism  stains  with  all  the  basic 

*  B 


"^ 


t 


FIG.  62. — Forms  of  B.  diphtheria  in  cultures -on  Loffler's  serum.  A, 
Characteristic  clubbed  and  irregular  shapes  with  irregular  staining  of  the 
cell  contents.  X  noo.  B,  Irregular  shapes  with  even  staining.  X  1000. 
(After  Park  and  Williams.) 

dyes,  notably  by  LofHer's  blue,  or  Neisser's  special  granule  stain. 
It  is  also  stained  by  Gram's  method.  The  length  of  the  organism 
differs  much,  according  to  the  reaction  of  the  medium  in  which  it 
grows.  Alkaline  media  favor  long  forms,  and  acid  the  reverse. 
Its  length  is  from  i-5/i  to  3.5^-  It  does  not  form  chains.  Bizarre, 
or  involution  shapes  predominate  in  old  cultures  (Fig.  63). 

Culture  and  Temperature  Requirements. — It  grows  best  at 
body  temperature,  and  on  glycerine  agar,  or  in  Loffler's  blood 
serum  mixture  of  alkaline  reaction. 

Vital  Characteristics— It  resists  drying  for  a  long  time,  and  has 


DIPHTHERIA  BACILLUS  2OQ 

lived  on  culture  media  for  eighteen  months  at  room  temperature; 
also  in  silk  threads  for  several  months  in  a  dried  condition. 
Remains  alive  in  healthy  throats  for  months.  Formalin  vapor 
kills  it  speedily;  corrosive  sublimate  solution,  i- 10,000,  destroys 
it  in  a  few  minutes;  light  is  lethal  to  it  in  from  two  to  ten  hours, 
and  heat  at  58°C.  in  ten  minutes. 

Habitat. — It  has  not  been  found  in  sewage,  or  sewer  gas,  soil 
or  water,  the  disease  therefore  is  never  transmitted  by  these  means. 


FIG.  63.— Diphtheria  bacilli  involution  forms.     (Kolle  and  Wassermann.) 

Has  been  found  in  the  throat,  nose,  and  in  the  conjunctivas  of 
healthy  bodies.  In  disease,  the  organism  is  mostly  found  in  the 
throat,  but  has  been  isolated  from  all  the  organs  in  some  fatal 
cases.  Sometimes  it  is  discovered  in  the  throats  of  animals. 
Though  its  action  is  local,  it  elaborates  a  toxin  which  acts 
systematically. 

Cultures. — On  gelatine  plate  the  growth  is  scanty  and  raised. 
This  medium  is  never  for  cultivating  this  organism.  The 
gelatine  is  not  liquefied.  On  glycerine  agar  plates  the  growth, 
though  moderate,  is  typically  characteristic,  but  very  slightly 

14 


210  BACTERIA 

raised  above  the  medium,  and  is  of  duller  lustre.  Old  colonies 
become  yellowish-brown,  the  center  of  which,  under  a  magnifica- 
tion of  sixty  diameters,  appears  darker,  and  with  ravelled  edges. 
On  Lb'ffler's  blood  serum  mixture,  the  organism  grows  rapidly 
and  well.  This  and  ascites-glycerine-agar  culture  media  are 
the  best  for  it.  Bouillon  made  from  fresh  meat  is  an  excellent 
medium  for  its  growth.  The  bouillon,  which  must  be  alkaline 
and  freshly  made,  becomes  first  cloudy;  then  a  fine  precipitate 
settles,  and  over  the  surface  a  delicate  pellicle  forms.  The 
reaction  of  the  culture  presents  three  types:  A,  is  acid  in  the 
beginning,  and  becomes  progressively  more  acid.  B,  is  alkaline 
from  the  start,  and  progressively  more  alkaline;  this  is  the  most 
toxic  growth.  C,  acid  at  the  start,  becoming  alkaline  finally. 
The  growth  is  not  so  luxuriant  as  in  B,  nor  is  there  as  much 
toxin  produced.  In  milk,  the  growth  is  luxuriant,  without 
coagulation.  The  reaction  is  amphoteric,  but  in  old  cultures  it 
becomes  alkaline.  On  potato,  rendered  alkaline,  it  will  grow, 
but  not  characteristically. 

Chemical  Activities. — No  gas  is  formed,  or  any  curdling  or 
gelatine  dissolving  ferment.  Acids  are  evolved  from  sugars; 
even  the  sugar  found  in  meat  is  converted,  into  lactic  acid.  In 
the  manufacture  of  toxin  this  muscle  sugar  must  be  removed.  A 
soluble  toxalbumin  is  created,  both  in  the  body  and  in  culture, 
which  is  intensely  poisonous.  See  chapter  on  bacterial  products. 

Pathogenesis. — Diphtheria  in  man  rneans  generally  an  infec- 
tion of  the  mucous  membrane  of  the  upper  respiratory  tract,  with 
the  formation  of  false  membranes.  The  latter  may  cause  death 
by  suffocation.  Infection  may  occur  in  the  skin,  vagina,  or  pre- 
puce. The  toxin  not  only  causes  a  local  necrosis,  with  the  forma- 
tion of  an  exudate,  consisting  of  fibrin  and  leucocytes,  but  also 
grave  systemic  action,  with  marked  degeneration  of  important 
nerves  and  nerve  centers,  and  also  of  the  parenchyma  of  the 
kidneys,  liver,  and  heart,  paralysis  following.  In  certain  struc- 
tures fragmentation  of  the  nuclei  of  the  cells  is  noted.  Guinea 


DIPHTHERIA  BACILLUS  211 

pigs,  cats,  horses,  and  cows,  may  be  infected  artificially,  but  the 
disease  never  occurs  spontaneously  in  these  animals.  Horses, 
dogs,  and  cattle  are  susceptible  to  its  toxin.  Diphtheria  bacilli 
often  have  associated  with  them,  streptococci,  which  add  to  their 
virulence,  and  complicate  the  disease.  Endocarditis,  adenitis, 
pneumonia,  abscesses,  and  empyemia,  may  be  caused  by  them. 
There  may  be  puerperal  diphtheria,  due  to  the  infection  of  the 
puerperal  tract.  Diphtheria  is  spread  mostly  by  personal  contact 
with  individuals  suffering  from  the  disease,  or  with  convalescents, 
in  whose  throats  virulent  bacilli  linger,  perhaps,  for  months.  It 
may  originate  from  infected  milk,  contaminated  from  human 
sources. 

Perhaps  the  most  important  source  of  infection,  especially  dur- 
ing an  epidemic,  is  the  healthy  bacillus  carrier  who,  wholly  una- 
ware of  his  condition,  is  carrying  virulent  germs  in  his  throat. 
This  further  indicates  that  individual  resistance  or  susceptibility 
plays  an  important  part  in  infection.  (See  Schick  Test.) 

Immunity  is  natural,  active,  artificial,  or  passive.  Active  im- 
munity, following  infection,  is  seldom  permanent  for  although  the 
individual,  if  he  recovers,  may  be  considered  immune  for  a  time, 
some  individuals  are  more  susceptible,  and  suffer  several  attacks. 
In  active  immunity  anti-toxin  is  found  in  the  blood,  and  recovery, 
and  subsequently,  immunity  are  due  to  this  fact.  Anti-toxin 
may  be  discovered  in  the  blood,  by  mixing  it  with  toxin  of  known 
strength,  and  injecting  it  into  guinea  pigs.  If  these  survive  a 
large  lethal  dose  of  the  toxin,  it  is  safely  presumed  that  anti-toxin 
was  present  in  the  serum  abstracted. 

Passive  artificial  immunity  is  induced  by  injecting  anti-toxin  in 
the  bodies  of  persons  exposed  to  diphtheria.  It  is  most  effective 
but  is  short-lived,  lasting  only  a  few  weeks.  Serum  therapy  (see 
anti- toxin  in  previous  chapter).  If  there  is  one  natural  specific 
cure  for  any  disease,  it  is  diphtheritic  anti- toxic  serum,  which  is 
prepared  by  immunizing  horses  with  toxin,  and  abstracting  their 
blood.  The  earlier  it  is  given,  the  better  are  the  chances  of  recov- 


212  BACTERIA 

ery.  As  a  prophylactic,  from  600  to  1,000  units  should  be  used. 
As  many  as  100,000  units  have  been  injected  in  a  single  patient. 
No  case  is  too  trivial,  or  too  far  advanced  in  which  to  use  it.  The 
serum  is  anti-toxic,  and  not  bactericidal.  Wassermann  has  pre- 
pared a  serum  that  is  bactericidal,  and  is  designed  to  destroy  the 
bacilli. 

Pseudo-diphtheria  bacilli,  which  morphologically  and  culturally 
resemble  the  true  bacilli,  have  been  described.  They  are  not 
pathogenic,  in  the  sense  of  producing  exudative  diphtheria,  and 
are  believed  to  be  attenuated  diphtheria  bacilli  by  many  observ- 
ers. The  diagnosis  of  diphtheria  by  culture  is  an  important 
measure.  It  depends  upon  the  rapid  growth  of  the  bacilli  upon 
Loffler's  blood  serum.  Of  all  the  various  organisms  found  in  the 
throats  of  patients  with  diphtheria,  the  diphtheria  bacilli  outstrip 
them  in  rapidity  of  growth.  After  eight  to  twelve  hours,  the 
serum  inoculated  with  the  smear  from  the  false  membrane  is 
covered  with  fine  granular  colonies  of  pure  diphtheria  bacilli. 
After  twenty-four,  or  more  hours,  the  other  organisms  present 
overgrow  the  diphtheria  colonies.  A  sterile  swab  of  cotton,  or  a 
stick,  is  rubbed  over  the  false  membrane,  or  throat,  and  then  over 
the  serum;  the  latter  is  incubated,  and  the  culture  examined  after 
eight  or  twelve  hours,  by  staining  with  LorHer's  blue.  If  curved, 
clubbed,  irregularly  stained  bacilli  are  found,  especially  if  they 
contain  dark  polar  granules,  and  are  generally  uneven  in  size  and 
bizarre,  it  may  be  safely  considered  that  they  are  diphtheria 
bacilli.  Gram's  stain  may  be  needed  to  confirm  the  diagnosis 
occasionally,  or  it  may  be  necessary  to  inoculate  guinea  pigs. 
This  may  be  done  by  inoculating  the  whole  throat  culture  and 
plating  out  from  the  local  inflammation,  or  the  organisms  may  be 
isolated  directly  by  plating  and  then  injected  into  animals. 
It  is  well  to  check  up  the  test  by  immunizing  one  animal  with 
200  units  of  anti-toxin  before  the  culture  is  given,  using  another 
guinea  pig  unprotected.  This  serves  as  a  control  upon  toxin 
production.  Virulent  bacilli  will  kill  a  guinea  pig  of  250  grams 


PSEUDO-DIPHTHERIA  BACILLUS  213 

in  three  days  if  i  c.c.  of  forty-eight  has  serum  broth  culture  be 
given  subcutaneously. 

Certain  of  the  pseudo-diphtheria  bacilli,  or  "  diphtheroids "  as 
they  are  called,  seem  to  have  the  power  of  increasing  or  continuing 
inflammation  after  it  has  been  started  by  other  germs.  They  are 
frequently  found  in  sinusitis,  prostatitis,  bronchitis,  etc.  In  a  few 
reported  cases  they  seem  to  have  been  the  only  micro organismal 
cause  of  disease  but  their  pathogenic  powers  are  usually  not  great. 

PSEUDO-DIPHTHERIA  BACILLUS 

Corynebacterium  Pseudo-diphtheriticum. 

Pseudo-diphtheria  Bacillus  (Hoffmann). 

Morphology  and  Stains. — This  bacillus  resembles  the  diph- 
theria bacillus.  The  rods,  however,  are  shorter  and  thicker; 
otherwise,  it  stains  like  the  true  bacillus,  but  not  by  Neisser's 
method. 

Culture. — On  glycerine  agar  the  growth  becomes  diffuse, 
spreading  from  the  line  of  inoculation  in  a  grayish-yellowish 
pasty  expanse.  It  grows  well  on  gelatine.  In  bouillon  it  forms 
a  denser  and  more  luxuriant  growth  than  the  bacillus. 

Habitat. — It  is  found  in  healthy  throats  and  conjunctivas. 

Pathogenesis. — It  is  non-pathogenic  for  guinea  pigs  (see  above). 

Diagnosis. — It  can  be  differentiated  from  the  true  bacillus  by: 

1.  Being  non-pathogenic. 

2.  Not  exhibiting  polar  granules  with  Neisser's  stain. 

3.  Not  producing  acids  in  certain  carbohydrate  media. 
Bacillus  xerosis  is  a  pseudo-diphtheria  organism  found  on  the 

normal  conjunctiva.     It  is  not  thought  to  possess  any  virulence. 

TUBERCLE  BACILLUS 

Mycobacterium  Tuberculosis. 

Bacillus  tuberculosis  (Fig.  64). 
Tubercle  bacillus. 


214  BACTERIA 

Morphology  and  Stains. — Slender  rods,  generally  unbranched, 
i~5ju  long,  and  4/4  thick,  usually  slightly  bent;  are  non-motile, 
and  have  no  spores  or  flagella.  In  old  cultures,  and  sometimes 
in  sputum,  branching  forms  are  seen,  and,  rarely,  some  that  are 
club-shape.  On  acid  potato,  thread  forms  are  found.  In  the 
continuity  of  most  of  the  bacilli,  unstained  spaces  are  seen;  in 
others  dense  deep  red  granules  are  found 
by  fuchsin.  As  this  bacillus  is  difficult  to 
stain,  special  methods  have  been  devised  to 
demonstrate  it,  as  the  sheathing  capsule 
renders  it  extremely  unsusceptible  to  the 
ordinary  methods  of  staining.  The  cause  of 
this  resistance  is  supposed  to  be  a  fatty  or 

FIG.  64. — Tubercle  waxy  substance  in  the  capsule  which  is  more 
bacilli  in  sputum:  ,  ,  ,  ,  ,  r  i  <•  ,  .,1  *  • 

stained  with  fuchsin    than  probable,  because  of  the  fact  that  stains 

and  methylene  blue,    that  are  fat  selective,  such  as  Sudan  III, 
(Greene's       Medical        ,  t*      -m  i»      i  i    i  r     i    • 

Diagnosis.)  color  it  very  well.     Boiling  hot  carbol-fuchsm 

gives  it  the  best  stain.  It  keeps  the  color 
in  spite  of  the  action  of  strong  solutions  of  mineral  acids  in 
water,  or  dilute  alcohol.  So  when  tissues,  or  secretions,  are 
stained  with  hot  carbol-fuchsin  for  a  short  time,  or  cold  carbol- 
fuchsm  for  a  long  time,  and  then  treated  with  a  25  percent 
solution  of  HNOa,  or  H2SO4,  in  water,  everything  is  deprived  of 
the  red  color,  except  the  tubercle  bacilli.  All  such  organisms 
that  are  acid  proof,  are  called  " acid-fast."  There  are  many 
other  bacilli  that  have  this  property.  Aniline  water  and  gentian 
violet  solution  also  stain  it.  Gram's  method  dyes  the  organism 
violet.  Sometimes  very  young  bacilli  do  not  stain  at  all. 

Vital  Requirements. — This  bacillus  thrives  best  at  37.5°C. 
It  grows  slowly,  is  a  strict  parasite,  and  an  obligate  aerobe.  In 
cultures  it  dies  quickly  in  sunlight,  and  in  diffuse  daylight  it 
dies  in  a  few  days.  It  resists  drying  and  light  in  sputum  for 
months.  Its  thermal  death-point  (moist)  is  80° C.  for  ten  min- 
utes; can  resist  6o°C.  for  one  hour,  but  succumbs  to  05°C.  in  one 


TUBERCLE  BACILLUS  215 

minute.  It  is  quickly  killed  by  formaline  and  corrosive  subli- 
mate, but  resists  3  percent  solution  of  carbolic  acid  for  hours. 
In  sputum  it  withstands  antiseptics  for  a  long  time. 

Chemical  Activities. — It  grows  slowly,  producing  no  coloring 
matter;  yields  an  aromatic  sweetish  odor,  but  no  gas  or  acid.  It 
produces  certain  plasmins  or  endo-toxins,  which  are  called 
tuberculins  (q.v.). 

Chemically  the  tubercle  bacillus  contains  two  fatty  matters, 
one  combined  with  an  alcohol  to  form  a  wax.  It  has  also  a 
protamin,  a  nucleic  acid  or  an  albumose.  Various  fatty  acids 
are  to  be  derived  from  it  by  chemical  treatment.  The  active 
principle  in  tuberculin  centers  around  its  protein  elements,  but 
is  not  exactly  known. 

Habitat. — It  is  a  strict  parasite  and  never  leads  a  saprophytic 
existence.  Is  found  wherever  human  beings  live  in  crowded 
quarters;  in  dust  of  rooms,  vehicles,  and  streets;  and  often  in 
milk  and  butter.  It  is  very  widely  distributed,  being  found  in  all 
human  communities. 

Cultures. — Since  the  organism  does  not  grow  below  3o°C., 
gelatine  is  never  used.  On  coagulated  blood  serum  of  cows, 
horses,  and  dogs,  this  bacillus  grows  best.  As  it  is  very  difficult  to 
isolate  in  pure  cultures,  the  following  procedure  should  be  followed : 
The  suspected  sputum,  fluid,  or  tissue  is  injected  into  a  guinea 
pig,  and  when,  in  two  weeks  or  more,  large  swollen  glands  can  be 
felt  in  the  groin,  the  animal  should  be  killed,  and  a  gland  removed 
under  strict  aseptic  precautions.  It  is  then  divided,  and  the 
halves  containing  the  bacilli  are  rubbed  over  the  surface  of 
coagulated  dog  serum  and  allowed  to  remain  in  contact  with  it. 
The  serum  should  be  coagulated  in  special  tubes,  with  glass  caps, 
having  small  perforations,  which  are  stopped  with  asbestos  fibre, 
or  glass  wool.  The  organism  grows  well  in  air,  but  too  great 
access  thereto  dries  and  kills  it.  After  the  tubes  are  incubated 
for  a  week  or  two,  little  scales  growing  unto  clumps  appear, 
which  are  lobulated  and  friable.  At  first  white,  it  later  turns 


216 


BACTERIA 


darker.  This  medium  is  never  liquefied 
by  the  culture.  On  glycerine  agar 
made  of  veal  broth  containing  6  per- 
cent of  glycerine,  the  organism  grows 
well  after  isolation  from  the  tissues, 
often  luxuriantly  (Fig.  65).  A  wrinkled 
film  covers  the  surface  of  the  agar, 
from  which  it  is  removed  with  ease. 
On  bouillon,  made  of  veal  and  glycer- 
inized,  it  develops  rapidly,  covering 
the  medium  with  a  dense  white 
wrinkled  pellicle,  which,  though  thick, 
is  friable.  After  a  time  it  falls  to  the 
bottom  of  the  flask.  It  grows  well  on 
glycerinized  potato  also,  and  milk 
agar.  On  egg-albumins  mixed  together, 
sterilized  and  coagulated,  this  bacillus 
also  develops  well. 

Pathogenesis. — The  discovery  of  the 
tubercle  bacillus,  its  methods  of  culti- 
vation and  differential  staining,  may 
be  ranked  with  the  greatest  of  medical 
discoveries.  This  organism  causes  in 
man  and  cattle,  chiefly,  the  disease 
called  tuberculosis.  It  rarely  attacks 
the  carnivora,  but  has  been  found  in 
such  animals  when  confined.  Swine 
are  often  infected;  cats  and  dogs  some- 
times, but  sheep,  goats,  and  horses 
seldom.  It  is  easy  to  inoculate  guinea 
pigs  or  rabbits  by  injection  or  feeding. 

FIG.  65. — Bacillus  tuberculosis;  glycerine 
agar-agar  culture,  several  months  old. 
(Curtis.) 


TUBERCLE  BACILLUS  217 

The  disease  is  widespread,  but  is  much  more  common  where 
human  beings  are  huddled  together  in  dark,  badly  ventilated 
rooms  and  shops.  In  tissues,  the  characteristic  lesion  is  a  tuber- 
cle. This  is  a  globular  mass,  about  the  size  of  a  very  small  shot, 
and  grayish  pearly  white.  Microscopically,  in  the  centre  of  the 
tubercle,  are  found  several  large  multinuclear  cells,  called  giant 
cells,  which  often  contain  thirty  or  more  nuclei,  and  a  number  of 
tubercle  bacilli,  the  nuclei  often  being  situated  at  one  pole,  while 


FIG.  66. — Tubercle  bacilli  showing  involution  forms.  (Kolle  and 
Wassermann.) 


the  bacilli  are  at  the  other.  About  the  giant  cells  epithelioid 
cells  are  grouped,  and  about  these  small  round  cells  are  massed  in 
great  numbers.  No  new  blood-vessel  formation  is  ever  found 
in  the  epithelial  cell  layers,  or  among  the  giant  cells.  Owing 
to  insufficient  blood-supply  the  centre  of  the  tubercle  frequently 
undergoes  caseous  degeneration.  If  the  lesion  heals,  the  caseous, 
centres  become  calcareous,  and  the  periphery  changes  into 
connective  tissue.  If  the  tubercles  coalesce,  great  masses  of 
caseous  tissue  form.  If  the  latter  becomes  infected  with  other 


2l8  BACTERIA 

pathogenic  bacteria  (streptococci  and  pneumococci)  rapid  soften- 
ing occurs,  with  cavity  formation,  etc.  Tubercles  may  develop 
in  any  organ  or  tissue  of  the  body.  The  lungs,  intestines, 
peritoneum,  glands,  larynx,  spleen,  and  bones  become  infected. 
The  liver  and  pancreas  seem  to  resist  invasion  more  than  other 
organs.  Bacilli  are  rarely  found  in  the  blood  in  tuberculous 
diseases.  They  may,  however,  be  found  in  the  urine,  in  kidney 
or  bladder  tuberculosis.  Milk  from  tuberculous  cows,  with, 
infected  udders,  often  contains  bacilli,  and  is  certainly  a  means 
of  transmitting  the  disease.  Cerebro-spinal  fluid,  in  tuberculous 
meningitis  contains  the  bacilli.  Bacilli  may  penetrate  mucous 
membranes,  and  not  causes  any  local  lesions,  but  infect  distant 
organs.  Tuberculosis  may  be  spread  in  the  body  in  four  ways. 
Sputum  may  be  swallowed  and  infect  the  intestines,  by  continuity, 
by  the  lymph-stream,  or  by  the  blood;  this  may  cause  intestinal 
ulceration  and  invasion  of  the  peritoneum.  If  the  bacilli  reach 
the  blood-stream,  the  disease  produced  is  generally  acute  miliary 
in  type.  This  is  manifested  by  the  formation  of  fine  gray 
tubercles.  In  tuberculosis  of  the  lungs  it  is  more  than  probable 
followed  skin  inoculation,  either  by  accidental  or  intentional 
trauma  that  the  bacilli  are  inhaled.  Local  tuberculosis  has  often 
Tuberculous  mothers  may  have  tuberculosis  of  the  genital  tract, 
and  fathers,  having  tuberculous  testes,  discharge  bacilli  in  the 
semen.  Placental  transmission  of  the  bacilli  from  mother  to  child 
occurs  rarely. 

Types  of  Tubercle  Bacilli. — It  has  been  considered  probable  by 
many  observers  that  there  are  two  types  of  bacilli,  a  human  and  a 
bovine  type.  Theobald  Smith  was  the  first  to  advance  this 
theory.  Koch  has  announced  that  the  two  types  were  totally 
different,  and  that  the  human  was  incapable  of  infecting  cattle, 
and  the  bovine  was  not  pathogenic  for  man.  In  view  of  the  fact 
that  cattle  are  frequently  tuberculous,  and  the  bacilli  are  often' 
found  in  the  milk,  it  is  important  to  know  if  the  bovine  type  can 
develop  in  man.  Ravenel  has  shown  that  it  is  undoubtedly 


TUBERCLE   BACILLUS  2IQ 

pathogenic  for  human  beings.  Men  have  been  infected  on  the 
hands,  while  performing  autopsies  on  tuberculous  cattle,  and 
their  skin  lesions  showed,  histologically,  unmistakable  tubercles. 
Cattle  have  been  infected  by  bacilli  of  the  human  type.  The 
bovine  type  of  bacillus  differs  from  the  human  in  the  following 
ways: 

1.  It  is  much  more  pathogenic  for  guinea  pigs  and  rabbits. 

2.  It  produces  more  extensive  lesions  in  cattle. 

3.  It  is  shorter  than  the  human. 

4.  It  produces  more  alkali  in  acid  media. 

5.  It  is  more  readily  isolated  from  original  lesions  and  does 
not  demand  animal  juices  in  culture  media  so  emphatically. 

The  subject  of  the  infectiousness  of  bovine  tuberculosis  for  man 
has  lately  been  exhaustively  studied  by  Park  and  Krumwiede. 
Their  conclusions  are  that  bovine  tuberculosis  is  practically  a 
negligible  factor  in  adults.  It  very  rarely  causes  pulmonary 
tuberculosis  or  phthisis,  which  disease  causes  the  vast  majority 
of  deaths  from  the  spread  of  virus  from  man  to  man.  In  children, 
however,  the  bovine  type  of  tubercle  bacillus  causes  a  marked 
percentage  of  cases  of  cervical  adenitis  leading  to  operation,  tern 
porary  disablement,  discomfort  and  disfigurement.  It  causes  a 
large  percentage  of  the  rarer  types  to  alimentary  tuberculosis 
requiring  operative  interference  or  causing  the  death  of  the  child 
directly  or  as  a  contributing  cause  in  other  diseases.  In  young 
children  it  becomes  a  menace  to  life  and  causes  from  6L£  to  10 
percent  of  the  total  fatalities  from  this  disease. 

It  is  not  always  easy  to  differentiate  the  tubercle  bacillus  from 
other  pathogenic  and  comparatively  harmless  acid-fast  bacilli. 
Among  these  are  the  B.  lepra,  the  B.  smegmatis,  and  a  number 
of  organisms  found  in  butter,  milk,  hay,  grass,  and  in  the  blind 
worm.  Culturally,  the  difference  is  great.  The  surest  way  to 
differentiate  the  tubercle  bacillus  from  other  acid-fast  organisms 
is  by  animal  inoculations. 

For  the  discovery  of  tubercle  bacilli  in  materials  apt  to  contain 


220  BACTERIA 

other  acid-fasts  several  method  are  now  employed.  The  material 
to  be  examined  may  be  stained  in  the  ordinary  manner  and  then 
decolorized  by  Pappenheim  solution  or  a  saturated  solution  of 
methylene  blue  in  absolute  alcohol.  Preparations  should  be  dried 
thoroughly  before  using  such  solutions.  For  " enriching"  in 
organisms,  the  bulk  of  material,  e.g.,  sputum,  is  suspended  in  15 
percent  antiformin  (the  proprietary  name  for  a  mixture  of  Javelle 
water  and  caustic  soda),  allowed  to  stand  in  the  incubator  for  a 
while  and  the  suspension  centrifuged.  In  the  sediment  many 
more  bacilli  will  be  found  than  in  the  same  bulk  of  the  raw  speci- 
men. This  antiformin  seems  to  dissolve  mucus,  tissue  and  all 
bacteria  except  tubercle  bacilli.  The  method  can  be  used  to 
procure  cultures. 

Even  with  this  method  organisms  escape  detection  in  some 
certainly  tuberculous  lesions.  This  is  said  to  be  due  to  non-acid- 
fast,  but  Gram-staining  granules.  They  are  said  to  be  found  by 
a-modified  Gram-Weigert  staining,  according  to  Much.  Such 
specimens  should  always  be  injected  into  guinea  pigs  for 
corroboration. 

Immunity. — It  is  possible  to  immunize  cattle  against  virulent 
bovine  tubercle  bacilli  by  inoculating  them  previously  with  a  cul- 
ture of  human  tubercle  bacilli  that  have  been  grown  for  some   , 
time  on  culture  media,  and  thus  attenuated.     The  tuberculins,  J; 
if  injected  into  a  person  with  chronic  tuberculosis,  stimulate  the 
tissues  to  a  slightly  greater  resistance  to  the  disease.     Thus  far  • 
anti-tuberculous  sera  are  not  of  a  pronounced  or  certain  thera- 
peutic value.     By  immunizing  horses,  Maragliano  obtained  a 
serum  that  he  claims  is  effective.     The  milk  from  immunized 
cattle  is  used  as  a  diet  in  tuberculous  patients  by  him.     The  vari 
ous  tuberculins,  some  containing  endo-toxins,  or  plasmins,  in 
solution,  are  capable  of  stimulating  the  formation  of  agglutinins  in 
the  sera  of  man  and  animals.     Blood  from  infected  individuals 
also  contains  these  bodies.     The  agglutination  test  does  not  seem 
to  be  of  great  practical  diagnostic  value,  while  the  complement 


t 


BACILLUS  .OF   LEPROSY  221 

fixation  gives  some  information  and  is  growing  in  favor  as  assist- 
ance in  obscure  clinical  cases. 

BACILLUS  OF  LEPROSY 

Mycobacterium  Lepra.    Hansen. 

Lepra  Bacillus. 

The  original  description  is  of  a  pointed,  curved,  acid  fast  rod 
occurring  in  groups  within  lepra  cells.  In  recent  years  several 
different  organisms  have  been  isolated  on  media  containing  trypto- 
phan.  They  have  a  few  features  in  common:  frankly  acid 
fast  or  decolorized  with  difficulty.  Gram  positive,  non-motile, 
non-spore  forming,  staining  shows  beading  or  barring,  polar 
bodies,  all  capable  of  pleomorphism.  They  have  been  grouped 
into  4,  (i)  acid  fast  bacilli  varying  from  coccoid  to  filamentous 
shapes,  not  easily  isolated  but  growing  well  after  once  accustomed 
to  media;  growth  yellow  or  orange;  (2)  acid  fast,  non-chromogenic, 
plump  bipolar  rods,  short  and  long,  growing  with  great  sparsity 
on  laboratory  media;  (3)  diphtheroid  bacilli  staining  solidly,  or 
beaded,  growing  best  at  37°C.  in  a  yellow- white  manner  on  agar 
and  with  a  pellicle  on  broth;  (4)  anaerobic  bacilli  of  more  solid 
staining  character  and  growing  feebly  as  a  dry  band  on  media. 
Very  marked  variations  in  luxuriance  and  color  production  are 
noted  on  different  media. 

To  cultivate  the  leprosy  organisms  bits  of  tissue  are  stripped 
off  and  allowed  to  digest  with  trypsin  on  blood  serum  or  agar 
plates.  When  the  tissue  has  softened  and  the  bacilli  multiplied, 
transfers  are  made  to  serum  glycerine  media  or  those  containing 
tryptophan.  It  is  best  alkaline  in  reaction. 

Pathogenesis. — The  bacilli  are  seen  in  enormous  numbers  in 
lepra  cells  and  elsewhere  in  diseased  tissues  and  have  been  found 
in  the  blood.  The  lepra  cells  are  large  and  vacuolated,  and  literally 
crammed  full  to  bursting  with  bacilli.  In  general  the  leprous 

NOTE. — Tubercle  bacilli  causing  avian  and  fish  tuberculosis,  and  other  acid  fast  bacilli 
exist,  but  not  being  pathogenic  for  man,  are  not  described  here. 


222  BACTERIA 

lesion  resembles  a  tubercle,  as  it  consists  of  giant  cells,  epithelial, 
and  round  cells. 

Immunity. — There  is  very  little  accurate  knowledge  as  to 
immunity  against  this  organism;  of  late  bacterins  have  been  tried 
with  some  success  it  is  claimed. 

STREPTOTHRIX  (Eppinger)  OR  NOCAKDIA 

The  genus  of  truly  branching  mycelium-forming  higher  bacteria 
(see  page  3),  such  as  the  actinomyces,  belonging  to  the  group 
between  bacteria  proper  and  the  moulds,  called  Trichomycetes. 
Kruse  has  described  nineteen  different  members  of  the  strepto- 
thrix,  some  pathogenic  to  man  and  animals. 


FIG.  67. — Streptothrix  Candida.     (Kolle  and  Wassermann.) 

A  number  of  cases  of  streptothrix  (Streptothrix  Hominis)  infec- 
tion in  man  have  been  reported.  The  disease,  in  general,  resem- 
bles phthisis.  In  the  pus,  sputum,  and  stained  sections  of  these 
cases,  strep  to  thricial  threads  have  been  found  (Figs.  67  and  70). 

Morphology  and  Stains. — Threads  are  thick  and  short,  or  long 
and  slender,  depending  upon  the  medium  on  which  they  grow.  In 
bouillon  the  threads  are  thin  and  long,  on  blood  serum,  short  and 


STREPTOTHRIX  223 

thick.  When  stained  there  is  distinct  beading  and  fragmentation 
of  the  protoplasm. 

There  is  true  branching  of  an  irregular  type,  which  is  best  seen 
in  liquid  media.  These  threads  often  produce  spores  on  culture 
media.  The  threads  often  disappear  in  old  cultures,  leaving  only 
the  spores,  which  stain  with  carbol-fuchsin  and  do  not  decolorize. 
The  threads  stain  by  Gram's  method,  and  Gram-Weigert  method. 
The  threads  are  not  acid-fast. 

Vital  Characteristics. — These  organisms  live  for  years  in  cul- 
ture media  after  it  is  dry.  Spores  resist  dry  heat  at  6o°C.  to 
7o°C.  for  an  hour;  moist  heat,  6o°C.  however,  kills  them  after  an 
hour.  It  is  a  strict  aerobe. 

Cultures. — On  Loffler's  blood  serum,  according  to  Tuttle,  this 
organism  grows  slowly  in  whitish  colonies,  which  finally  become 
yellow.  The  adult  colonies  adhere  to  the  serum.  On  agar  it 
grows  rapidly  and  characteristically.  The  colonies  are  yellowish- 
white  and  adhere  to  the  agar.  In  Bouillon. — It  develops  slowly 
on  the  surface  of  the  medium.  Fluffy  tufts,  or  balls,  are  formed, 
that  sink  to  the  bottom  of  the  tube.  The  growth  is  whitish. 

Pathogenesis. — For  rabbits  and  guinea  pigs  this  organism  is 
pathogenic,  producing  abscesses,  tubercles,  induration,  etc.  It  is 
a  pus  forming  organism. 

In  animals  the  spontaneous  disease  appears,  best  known  as 
"farcin  du  boeuf,"  as  ulcerative  or  infiltrative  lesions  of  lungs 
and  skin. 

In  man,  the  disease  picture  is  like  that  of  tuberculosis.  It 
causes  abscesses,  adenitis,  indurations  of  the  skin,  endocarditis, 
and  pleuritic  inflammation.  Many  grayish  tubercles  were  found 
that  resembled  the  lesions  produced  by  the  tubercle  bacillus. 
Cavity  formation  has  been  described. 

This  organism  may  act  as  a  secondary  infecting  agent  in  tuber- 
culosis of  the  lungs.  Tuttle  reviews  twelve  cases,  all  of  which 
were  fatal. 

In  examining  sputum  from  tuberculous  cases,  in  which  the  typ- 


224 


BACTERIA 


ical  bacilli  are  not  found,  it  is  well  to  look  for  the  streptothrix  by 
staining  with  Gram's  stain. 

RAY  FUNGUS 

Actinomyces  Bovis. 

Ray  Fungus. 

Morphology  and  Stains. — This  organism  is  called  the  ray  fungus 
because  of  the  stellate  arrangement  of  its  threads  in  the  colonies 


FIG.  68. — Actinomyces  bovis.     (Williams..) 

found  in  tissues.  It  is  of  a  more  complex  structure  than  the  bac- 
teria hitherto  described.  There  are  three  elements  found  in  every 
colony:  (i)  long  thread  which  may  be  branched  or  unbranched; 

(2)  threads  that  are  clubbed,  which  may,  or  may  not,  be  branched; 

(3)  spore-like  bodies  contained  within  the  thread,  which  seem  to 
arise  by  breaking  up  of  the  threads.     The  colonies  in  tissues  are 
often  i  mm.  in  diameter,  and  made  up  of  many  clubbed-shaped 


RAY   FUNGUS 


225 


threads  radially  situated.  Through  the  periphery  and  extending 
beyond  are  other  unclubbed  threads,  while  scattered  throughout 
the  colony  and  beyond  it,  and  in  the  threads,  may  be  seen  many 
spore-like  bodies.  The  threads  and  spores  stain  by  Gram's 
method  while  the  clubs  do  not.  Basic  stains  also  color  all  the 
elements.  The  spores  do  not  stain  like  bacterial  endo-spores. 

Vital  Requirements. — It  is  a  facultative  anaerobe,  and  grows 
best  in  the  absence  of  air,  at  37°C.  Resists  drying  for  a  long  time, 
and  its  thermal  death-point  is  8o°C.  after  fifteen  minutes'  exposure. 


FIG.  69. — Actinomyces.     (Williams.) 

Chemical  Activities. — Slowly  liquefies  gelatine,  does  not  curdle 
milk;  and  produces  a  mouldy  odor.  No  gas  or  acids  are  formed, 
nor  is  H2S  developed. 

Habitat. — It  has  been  found  in  straw  and  hay,  but  never  in  a 
healthy  body. 

Cultures. — On  gelatine  plates  it  produces  yellowish-gray  colo- 
nies that  are  very  small.  These  grow  into  the  gelatine,  slowly 
liquefying  it.  The  colonies  are  very  tough  and  fibrous.  In  agar 
tubes  it  grows  very  slowly,  the  first  growth  being  like  dewdrops; 
later  these  enlarge,  turning  yellow,  and  finally  brown.  The  cul- 

15 


226  BACTERIA 

ture  grows  down  into  the  agar,  and  the  medium  darkens.  Old 
cultures  'are  dark  and  crumbly  looking,  adhere  firmly  to  the  agar, 
and  have  a  downy  dust-like  covering.  On  blood  serum  the  colo- 
nies appear  as  dewdrops,  which  later  become  brownish,  then, 
yellowish-orange,  or  brick-red.  In  bouillon  the  growth  is  at  the 
bottom  in  ball-like  masses  that  cohere  firmly.  Clubs  do  not  form 
in  this  medium.  The  supernatant  bouillon  is  clear,  with  no  sur- 
face growth.  In  milk  it  produces  no  chemical  change.  On 
potato  it  grows  in  knot-like  colonies. 

Pathogenesis. — Causes  in  cattle  the  disease  known  as  "lumpy 
jaw."  The  fungus  reaches  the  jaw  from  the  teeth  and  gums,  the 
latter  first  being  injured  by  sharp  spines  in  the  food.  In  man,  the 
internal  organs,  lungs,  intestines,  and,  rarely,  the  brain  become 
infected.  The  liver  often  is  abscessed.  In  both  cattle  and  man 
universal  actinomycosis  sometimes  occurs.  The  lesions  produced 
are  rather  massive  at  times;  the  nidus  is  often  surrounded  by 
enormous  numbers  of  polynuclear  leucocytes,  which,  no  doubt, 
play  a  defensive  role  in  the  tissues.  The  disease  is  often  fatal  to 
cattle  and  to  man.  It  is  hard  to  inoculate  laboratory  animals 
with  the  disease,  though  Wright  succeeded  in  so  doing.  No 
useful  immunity  reactions  seem  to  occur.-  Vaccines  have  been 
used  in  treatment  with  encouraging  results  but  no  cures.  Potas- 
sium iodide  internally  is  always  indicated. 

ACTINOMYCES  MADURA 

Actinomyces  Madura. 

Streptothrix  Madura,  Vincent. 

Morphology  and  Stains. — A  non-motile,  non-flagellated  organ- 
ism said  to  have  spores.  Its  growth  resembles  that  of  A  ctinomyces 
boms.  It  consists  of  long  threads  that  are  clubbed.  These  stain 
by  all  the  basic  aniline  dyes  and  by  Gram's  method.  There  are 
three  recognized  forms  of  this  organism,  white,  black  and  red, 
which  have  been  found  in  various  cases  but  the  interrelation  of 


ACTINOMYCES   MADURA  227 

which  is  not  yet  fully  understood.    The  description  given  is 
generally  applicable  only  slight  variations  being  noted. 

Vital  Requirements. — It  is  a  facultative  aerobe.  The  thermal 
death-point  for  the  spores  is  85°C.  for  three  minutes,  and  75°C. 
for  five  minutes.  Vegetative  thread  forms  die  at  6o°C.  Grows 
best  at  37°C.,  and  scantily  at  room  temperature. 


FIG.  70. — Streptothrix  hominis.     (Kolle  and  Wassermann.) 

Cultures. — Generates  upon  all  culture  media.  In  Bouillon. — It 
appears  in  little  clumps  which  cling  to  the  glass,  but  eventually 
sink  to  the  bottom  in  masses.  In  Gelatine. — It  grows  sparingly 
in  clumps,  slowly  liquefying  the  medium.  Upon  Agar. — It  forms 
shiny  round  colonies,  that  are  first  devoid  of  color.  They  resem- 
ble an  umbilicated  vaccine  vesicle  and  adhere  tightly  to  the  agar. 
In  Milk. — It  grows  without  coagulating  the  medium.  On 
Potato. — The  culture  is  very  slow,  and  without  chromogenesis. 
Old  colonies  are  powdery,  due  to  spores. 

Pathogenesis. — In  man  it  produces  madura  foot,  an  affection 
characterized  by  induration,  ulceration,  and  fistulas  formation 
with  pus. 


228  BACTERIA 

BLASTOMYCOSIS 
OIDIOMYCOSIS 

Oidium  Albicans.  Thrush,  Soor. — This  organism  resembles 
both  a  yeast  and  a  mould,  because  it  exhibits  characteristics  that 
are  common  to  both  of  these  forms.  It  exhibits  budding  yeast 
cells  and  budding  mycelia.  The  yeast  cell  is  6ju  long  and  i/*  wide, 
but  the  cells  vary  very  much  in  length  and  width. 

It  stains  well  in  tissues  and  cultures  by  Gram's  method,  and  by 
the  ordinary  basic  stains.  It  may  be  cultivated  on  bouillon, 


FIG.  71. — Thrush  fungus.     (Kolle  and  Wassermann.) 

blood  serum,  agar,  potato,  etc.,  and  it  is  rather  indifferent  to  the  ; 
reaction  of  the  media.     It  grows  best  if  sugars  are  present.    It  is,  j 
however,  very  susceptible  to  such  antiseptics  as  phenol,  salicylic 
acid,  subMmate,  etc. 

Pathogenesis. — Causes  in  man  a  condition  known  as  oidio-  , 
mycosis,   and  in  young  children  a  very  troublesome .  stomatitis,  : 
which,  if  the  child  is  weak  and  illy  nourished,  may  result  seriously. 
It  may  cause  metastatic  abscesses  in  the  brain,  spleen,  and  kid- 


OIDIOMYCOSIS 


229 


neys,  or  nodules  in  the  lungs.     This  organism  may  penetrate- 
mucous  tissues,  and  fill  the  lumen  of  vessels  (Virchow). 

Tropical  spruce  is  held  by  some  as  due  to  a  near  relative 
of  the  oidium,  namely  Monilia  psilosis.  The  organism  is  to  be 
found  all  along  the  alimentary  tract.  It  possesses  many  cultural 
characters  like  oidium.  Vaccines  are  said  to  be  of  practical  use 
in  therapeutics. 


FIG.  72. — Doubly  contoured  organisms  found  in  oidio mycosis  (blastomyco- 
sis).     (From  Buschko  after  Hyde  and  Montgomery.) 

Sporothricosis  is  a  subacute  or  chronic  infection  usually  of  the 
skin,  but  at  times  involving  the  internal  organs,  caused  by  the 
Sporothrix  Schencki.  The  organisms  grow  as  delicate  mycelia 
with  very  numerous  spores,  2-4  X  3~6/i  in  size.  They  grow  best 
upon  acid  media  at  body  temperature  as  white  fluffy  masses 
which  later  become  brown.  The  disease  in  man  takes  the  form 
of  firm  tumefactions  under  the  skin  which  may  ulcerate  leaving 


230  SACTERIA 

indolent  ulcers.  Fever,  prostration  and  emaciation  follow.  Rats 
are  susceptible.  An  agglutinin  appears;  this  may  assist  in 
diagnosis. 

Saccharomycetes  are  sometimes  pathogenic.  They  are  bud- 
ding fungi,  multiplying  by  splitting  off  the  bud  when  conditions 
are  favorable  for  active  growth  but  capable  of  intracellular  sporu- 
lation,  ascospores,  when  under  adverse  conditions.  They  usually 
have  a  rather  resistant  capsule,  sometimes  double.  Saccharo- 
myces  busse  or  hominis  is  capable  of  setting  up  in  man  a  cutane- 
ous and  subcutaneous  ulcerative  and  infiltrative  or  even  suppura- 
tive  lesion  which  may  last  for  a  long  time;  involvement  of  internal 
organs  can  occur.  Transmission  to  animals  is  difficult.  The 
organisms  are  from  3  to  30^,  round  or  elliptical,  rarely  forming 
mycelia  in  the  tissues.  They  stain  well  but  not  by  Gram's 
method.  They  are  best  seen  by  mixing  the  pus  with  a  caustic 
solution.  They  grow  under  aerobic  conditions  at  37°C.  upon  acid 
serum,  dextrose  or  maltose  agar  as  white  plaques  which  later 
become  wrinkled  and  velvety.  They  are  easily  killed. 

Coccidiosis  or  oidiomycosis  is  an  infection  very  similar  to  the 
foregoing  but  the  causative  organism,  Coccidioides  immitis,  differs 
from  Sac.  hominis  in  showing  intracellular  sporuiation  and  no 
budding. 

MOULDS  OR  HYPHOMYCETES 

These  are  the  next  higher  order  of  plant  algae  and  consist  of 
cells  which  can  elongate  to  threads,  dividing  by  intracellular 
sporuiation  or  by  the  development  of  reproductive  organs  which 
in  some  varieties  are  bisexual  in  character.  They  are  widely 
distributed  in  nature  living  mostly  as  saprophytes.  Diseases 
due  to  these  forms  are  practically  confined  to  the  skin  although 
extremely  rare  cases  of  dissemination  are  on  record. 

Ringworm  of  all  kinds  is  due  to  the  mould  Trichophyton  either 
of  the  species  megalosporon  or  microsporon.  The  spores  of  the 
former  are  7~8ju,  of  the  latter  2-3  ju.  They  grow  readily  as  dis- 


MOULDS  231 

crete  mammillated  fluffy  colonies.  They  consist  under  the  micro- 
scope of  slender  septate  hyphae. 

Favus  is  due  to  the  mould  Achorion  Schoenleinii.  This  fungus 
gives  off  hyphae  with  knob-like  reproductive  organs.  Spores  are 
oval  3~8/x  X  3~4ju.  This  fungus  grows  as  a  "scutulum"  on  the 
skin  eruption.  It  can  be  cultivated  on  sugar  agar,  as  a  waxy,  or 
downy  yellow  or  white  round  plate  with  a  central  mammillation. 

Pityriasis  versicolor  is  due  to  the  mould  Microsporon  furfur. 
It  is  similar  to  the  Trichophyta,  but  invades  only  the  superficial 
layers  of  the  skin. 

Aspergillus  Niger,  A.Fumigatus,  and  A.Flavus. — A  polycellular 
mycelial  organism  which  produces  spores  and  branched  threads, 
that  are  variously  named  from  the  macroscopic  appearances  of 
the  growth.  All  thrive  well  as  37°C.  and  may  be  cultivated  on 
the  usual  culture  media.  In  man,  the  external  auditory  meatus 
is  often  infected  with  these  organisms,  causing  a  troublesome  dis- 
ease. They  may  infect  the  lungs  of  weak  anaemic  subjects  with 
wasting  diseases,  and  may  be  pathogenic  for  cattle,  horses,  and 
birds. 

The  author  has  found  that  the  young  hyphae,  the  sporangiar 
and  spores  of  some  of  these  hyphomycetes  (moulds)  if  treated  with 
hot  or  boiling  alkaline  solution  of  copper  sulphate  are  stained  be 
the  copper,  which  has  an  affinity  for  them,  and  appear  a  light  lilac- 
blue  under  the  microscope.  If  treated  with  a  solution  of  ferric 
cyanide  of  potash  and  acetic  acid,  these  stained  parts  turn  a 
dark  brown,  showing  that  there  is  an  actual  absorption  or  per- 
haps chemical  union  of  the  protoplasm  of  the  mould  with  the 
copper.  Some  moulds  are  stained  a  deep  blue,  and  are  visible 
to  the  naked  eye  in  test-tubes,  after  treatment  with  the  boiling 
alkaline  copper  others  are  colored  a  bright  yellow.  Some  moulds 
and  bacteria  have  the  power  of  reducing  copper  in  Fehling's 
"solution. 


CHAPTER  IX 
ANIMAL  PARASITES 

While  numerous  diseases  are  caused  by  vegetable  parasites, 
such  as  bacteria  and  moulds,  there  are  others  in  which  the  etio- 
logical  role  is  played  by  minute  microscopic  organisms  of  the 
animal  kingdom.  There  are  also  infectious  diseases  that  are 
supposedly  caused  by  animal  parasites,  and  yet  the  exact  knowl- 
edge that  they  are  the  cause  is  lacking.  Not  all  of  the  pathogens 
of  the  animal  kingdom  will  fulfil  Koch's  postulates  but  their  num- 
ber is  increasing.  Within  the  past  few  years  it  has  been  found 
possible  to  cultivate  Trypanosomata,  spirochaetee,  amoebae,  and 
hemosporidia  with  completipn  of  Koch's  postulates  in  the  first  two. 

In  general,  it  may  be  said  of  animal  parasites,  particularly  those 
belonging  to  the  protozoa,  that  an  intermediate  host,  such  as  a 
suctorial  insect,  is  necessary  for  the  transmission  of  the  organism 
to  man  or  animal.  This  is  called  alternate  generation  and  is  a 
very  characteristic  feature. 

The  protozoa,  as  parasites  in  man,  are  the  cause  of  several  well- 
known  diseases,  namely:  dysentery,  malaria,  sleeping-sickness, 
and  coccidiosis.  In  hydrophobia,  scarlet  fever,  and  small-pox  cer- 
tain peculiar  bodies  are  constantly  found  that  resemble  protozoa, 
but  since  it  is  not  known  whether  they  are  animal  bodies  at  all, 
they  cannot  be  classed  as  protozoa,  and  are  discussed  under 
Chlamydozoa  in  the  next  chapter. 

PROTOZOA 

The  protozoa  of  importance  as  disease  producers  are  to  be  found 
in  the  classes,  orders  and  families  given  as  follows: 

232 


PROTOZOA  233 

Protozoa. 
Sarcodina. 

Rhizopoda. 

Amoebina — Amoebae. 
Mastigophora. 
Flagellata. 

Monadida,        Cercomonas,    Trypanosoma,    Poly- 

mastigida,  Trichomonas. 

Some  authors  separate  a  family  Spirochaetidae  to  in- 
clude Spirochaeta  and  Treponema. 
Sporozoa. 

Gr  egarinida — gr  egarines . 
Coccidia — coccidia. 
Hemosporidia. 

Plasmodium — malaria. 
Infusoria. 
Cilia  ta. 

Heterotrichida — Balantidium. 

The  protozoa  are  always,  in  every  stage  of  development,  primi- 
tive unicellular  bodies.  They  consist  essentially  of  a  cell  body  or 
sarcode,  a  nucleus,  and  a  nudeolus.  All  of  the  vital  functions  of 
the  cell  are  carried  out  by  the  cell  body,  the  protoplasm  of  which 
digests  and  assimilates  food.  Particular  parts  of  the  protoplasm 
have  special  functions,  these  parts  are  called  organdies.  The 
living  protoplasm  is  finely  granular,  is  viscid,  and  exhibits  a  dis- 
tinct movement.  The  motility  of  protozoa  is  supplied  variously. 
In  the  Rhizopoda  progression  takes  place  by  pseudopods  or  false 
feet,  a  phenomenon  in  which  a  section  of  the  cell  wall  and  proto- 
plasm are  extended  like  a  bud.  Into  this  the  latter  then  flows 
with  a  shrinkage  of  the  main  body.  At  last  the  pseudopod  is 
large  enough  to- hold  all  the  protoplasm  and  the  former  place  of  the 
protozoon  is  vacated  for  the  new.  Motility  is  also  supplied  by 
the  lashing  or  vibratory  action  of  flagella  or  the  fine  vibration  of 


234  ANIMAL   PARASITES 

circumferential  cilia.  In  others  a  special  muscular  segment 
of  the  body  may  exist.  The  suctorial  tubes  act  also  for  motion 
at  times.  In  most  protozoa  two  layers  can  be  seen — the  ectosaro, 
and  endosarc.  The  ectosarc  originates  the  movement,  is  concerned 
in  the  ingestion  and  excretion  of  food,  and  the  respiration.  The 
endosarc,  which  circulates  slowly,  is  mainly  for  digestive  purposes. 
In  it  are  ferments,  crystals,  food  particles  (seen  in  the  food  vacu- 
oles),  oil  globules,  gas,  and  pigment  granules. 

Flagella  and  suctorial  tubes — in  protozoa  that  have  them — 
belong  to  the  ectosarc.  Skeletal  tissues,  shells,  etc.,  also  belong 
to  this  layer. 

The  food  consists  of  bacteria,  smaller  animals,  algae,  and  animal 
waste. 

Propagation  is  effected  by  direct  cell  division,  beginning  in  the 
nucleus,  by  cell  budding  or  by  a  complicated  course  of  sporulation 
which  may  be  sexual  or  asexual.  Sometimes  division,  or  budding, 
occurs  rapidly  without  the  segments  separating,  leading  to  the 
formation  of  protozoal  colonies,  or  swarm  spores. 

In  the  case  of  the  malarial  plasmodia,  asexual  development, 
(schizogony)  takes  place  in  man's  blood,  while  the  sexual  develop- 
ment (sporogony)  takes  place  in  the  mosquito.  Protozoa  are 
found  in  salt  and  fresh  water,  in  damp  places,  and  in  animals  as 
parasites. 

Since  the  zoological  classification  has  been  given  and  may 
used  for  reference  to  larger  works,  the  various  pathogenic  proto- 
zoa are  given  separately  without  direct  reference  to  their  sys 
tematic  classification. 

There  are  but  two  Rhizopods  that  are  parasitic  and  pathogenic 
to  man.  The  only  one  of  these  of  any  import  is  the  Amoeba. 

AMCEBA  DYSENTERIC  OR  ENTAMCEBA 
HISTOLYTICA 

This  is  a  pear-shaped  roundish  body  from  .008  to  .05  mm.  ii 
diameter.  The  ectosarc  is  easily  discernible  in  the  pseudopodia, 


AMCEBA   DYSENTERIC  235 

but  not  in  the  round  quiescent  cell.  In  the  endosarc,  which  is 
granular,  vacuoles  are  easily  seen;  so  are  fragments  of  food,  red 
and  white  blood  cells,  bacteria,  eipthelial  cells,  and  faecal  matter. 
The  pseudopodia  are  broad  and  lobose;  one  or  two  are  protruded 
at  a  time.  The  motion  of  the  organism  depends  upon  the  reaction 
of  the  media,  and  the  temperature.  The  vacuoles  and  nucleus 
are  always  present.  Propagation  generally  takes  place  by  binary 
division,  the  process  beginning  in  the  nucleus.  When  irritated, 
the  amoeba  at  once  assumes  a  spherical  form,  the  pseudopodia 
being  withdrawn. 

Pathogenesis. — Amoeba  dysenteriae  is  the  cause  of  the  protozoal 
form  of  dysentery.  So  far  as  known  this  particular  variety  exists 
only  in  the  intestines  of  effected  persons.  Lesions  similar  to  those 
of  human  dysentery  have  been  produced  in  monkeys,  dogs  and 
cats,  and  the  amoebae  recovered  from  them.  Cultures  consisting 
only  of  amoebae  have  been  obtained  by  special  technique,  but  a  so- 
called  pure  mixed  growth  of  colon  bacilli  and  amoebae  is  cultivated 
with  little  difficulty.  In  the  lower  gut  of  man  and  cats,  in 
dysentery  cases,  encysted  amoebae  are  often  found.  They  have 
been  seen  in  the  liver  (in  old  cases),  also  in  the  lungs  and  sputum. 

Cats  have  been  infected  by  pus  from  liver  abscessed  devoid  of 
bacteria  (Kartulis).  The  urine,  in  cases  of  cystitis,  contained 
amoebae,  and  it  is  believed  to  be  the  cause  of  the  disease  in  some 
rare  instances.  In  dysentery  the  amoebae  are  the  cause  of  the 
necrosis  and  ulceration,  as  they  frequently  become  encysted  in 
the  submucous  tissues.  From  the  Entamceba  coli  the  dysenteric 
amoebae  is  differentiated  by  the  fact  that  it  is  larger,  coarser  in 
structure,  and  takes  up  red  blood  cells,  which  the  former  does 
not.  Differentiation  by  Wright's  stain  Entamceba  coli  ectoplasm 
light  blue,  endoplasm  dark  blue,  nucleus  red.  Ent.  histolytica 
ectoplasm  dark  blue,  entoplasm  light  blue,  nucleus  pale  red  or 
pink. 

Amoebae  quickly  loose  their  mobility  as  the  surrounding  tem- 
perature leaves  that  of  the  normal  body,  although  they  are  not 


236 


ANIMAL  PARASITES 


killed  at  low  temperatures,  and  resist  up  to  6o°C.  when  in  the 
encysted  state.  Quinine,  permanganate  of  potash,  weak  acid  and 
silver  nitrate  are  quickly  fatal  to  vegetative  forms.  Emetin  is 
a  useful  drug  since  it  kills  all  but  encysted  forms  after  a  very 
short  exposure  if  direct. 

Entamceba  tetragena,  formerly  classified  as  a  separate  variety, 
is  now  generally  believed  to  be  but  one  stage  in  the  development 
of  Ent.  histolytica. 

In  stools  (from  dysenteric  cases)  over  a  day  old,  amoebae  are 
not  often  found,  as  they  undergo  a  rapid  disintegration  outside 
the  body. 

Amoebae  are  cultivated  upon  stiff  agar  preferably  with  defibri- 
nated  blood  and  in  company  with  bacteria.  If  a  colony  can  be 
obtained  free  of  bacteria,  development  will  continue  on  agar 


FIG.  73. — Entam&ba  tetragena.     The  same  living  individual  drawn  at  brief 
intervals  while  moving.     (From  Doflein  after  Hartmann.) 


smeared  with  organ  extracts.  The  addition  of  dead  bacteria  to 
culture  media  seems  favorable  to  their  development.  The  poisoi 
is  not  known.  The  free  amoebae  in  the  colon  are  easily  killed,  bul 
when  encysted  are  more  resistant.  Quinine  is  fatal  to  cultures  ii 
ten  minutes  in  strength  of  1-2500.  Formalin  is  not  practicable. 
Endamoeba  buccalis  is  found  in  the  mouth  especially  in  carioi 


FLAGELLATA 


237 


teeth  and  in  inflamed  gums;  it  may  have  some  effect  in  con- 
tinuing a  gingivitis. 

FLAGELLATA 

The  flagellata  derive  their  name  from  the  fact  that  all  are  pos- 
sessed, at  some  time  in  their  existence,  of  flagella,  which  are  not 
only  organs  of  locomotion,  but  serve  to  apprehend  food. 

The  principal  members  of  this  class  of  interest  from  a  patho- 
logical viewpoint,  are  the  trypanosomes.  Trypanosoma  gam- 


FIG.  74.— Trypanosome  in  rats'  blood.     (Williams.) 

biense,  transmitted  by  the  tsetse-fly  Glossina  palpalis,  pathogenic 
for  man  (see  page  239).  The  Trypanosoma  brucei,  which  causes 
the  tsetse-fly  disease  (nagana)  in  horses  and  cattle,  is  transmitted 
to  cattle  by  the  bite  of  the  tsetse-fly,  Glossina  morsitans.  It  can 
be  grown  on  blood  agar  (Novy). 

Trypanosoma  evansi  causes  surra,  a  disease  of  horses  in  Central 
Asia  transmitted  by  a  fly  of  the  genus  Stomoxys. 


238  ANIMAL   PARASITES 

Trypanosoma  equiperdum  causes  a  sexual  disease  in  stallions 
and  mares  called  dourine;  this  is  akin  to  syphilis  in  man. 

Trypanosoma  lewisi  of  rats  is  transmitted  from  animal  to 
animal  by  means  of  fleas. 

Trypanosoma  noctuae. — A  parasite  of  the  little  owl,  which  is 
introduced  into  the  bird  through  the  bite  of  the  mosquito  Culex 
pipiens. 

Several  other  forms  have  been  noted  but  these  are  perhaps 
the  most  important  and  serve  as  examples  for  this  family  of 
protozoa. 

Trypanosomes  are  elongated  fusiform  bodies  pointed  at  both 
ends,  provided  by  a  fin  fold,  or  undulating  membrane,  running 
along  the  dorsal  edge  and  forming  frill-like  folds  which  terminate 
in  a  whip-like  extremity  or  flagellum. 

A  large  nucleus  is  always  seen,  also  a  centrosome,  a  small  chro- 
matic mass — likewise  called  a  blepharoplast — near  one  pole. 

The  flagellum  is  at  the  anterior  extremity;  the  blunt  pointed 
end  is  the  posterior  extremity.  Cell  division  begins  in  the  bleph- 
aroplast, the  cell  dividing  longitudinally,  the  nucleus,  flagellum, 
and  the  protoplasm  dividing  last.  Dividing  trypanosomes  fre- 
quently appear  in  clumps  with  the  ends. united,  resembling  a 
wheel. 

The  trypanosomes  exist  in  two  hosts — one  a  suctorial  insect — 
and  have  a  sexual  and  an  asexual  existence  (alternate  generation). 
In  an  infected  owl  the  organism  has  been  observed  clinging  fast 
to  the  red  cells,  absorbing  nutriment  during  the  day,  while  at 
night  it  swims  about  freely  in  the  plasma. 

In  owl's  blood  the  trypanosome  assumes  asexual  forms,  called 
macro  gametes.  These  macrogametes  penetrate  the  erythrocytes, 
accumulating  the  remnants  of  the  red  cells  in  the  protoplasm. 
The  nucleus  of  the  trypanosome^  may  be  seen  in  the  interior  of 
the  protoplasm.  The  microgametocytes  arise  from  the  asexual 
forms  and  when  mature,  give  rise  to  eight  microgametes. 


TREPANOSOMA   GAMBIENSE  239 

TRYPANOSOMA  GAMBIENSE 

Castellan!  found  that  this  trypanosome  is  the  cause  of  sleeping- 
sickness  among  the  natives  of  South  Africa,  and  the  organism  has 
been  found  quite  regularly  in  the  blood,  and  also  the  cerebro- 
spinal  fluid  sometimes  as  well,  in  this  disease.  The  disease  has  a 
long  period  of  incubation  (months),  runs  a  long  course  usually, 
and,  at  its  full  development,  it  is  a  meningo-encephalomyelitis. 
This  is  characterized  by  hebetude,  somnolence,  and  coma. 


FIG.  75. — Trypanosomes;  showing  ordinary  structural  appearance  on  left; 
in  middle  a  trypanosome  undergoing  division;  on  the  right  a  group  dividing 
in  radial  manner.  (Tyson's  Practice.) 

These  symptoms  are  accompanied  by  disturbance  of  the  motor 
apparatus,  oedema,  irregular  temperature,  rapid  pulse,  emaciation, 
skin  eruptions,  and  death  in  coma.  In  these  cases  the  parasites 
may  be  seen  in  the  blood  slowly  winding  their  way  through  the 
corpuscles.  The  pathogenic  action  is  due  no  doubt  to  some 
toxin  elaborated. 

The  disease  is  transmitted  from  man  to  man  by  the  tsetse-fly 
(Glossina  palpalis).  In  the  fly  it  exists  as  a  true  parasite  in  a 
host,  and  not  merely  passively.  It  becomes  infective  within 
three  days  of  biting  and  remains  so  for  four  weeks. 

The  disease  does  not  depend  upon  the  age,  sex  of  the  individual, 
nor  upon  drinking  water,  food,  seasons,  etc. 

The  organism  may  be  stained  by  the  ordinary  blood  stains, 


240  ANIMAL   PARASITES 

mixtures  such  as  Irishman's,  Romano  wsky's,  etc.,  the  nucleus, 
centrosome  and  flagella,  staining  deepest.  Thus  far  the  T.  gam- 
biense  has  not  been  cultivated  in  artificial  media. 

Novy  has  succeeded  in  growing  the  T.  lewisii  and  T.  brucei  on 
agar  mixed  with  defibrinated  rabbit's  blood.  These  are  the  first 
animal  parasites  to  be  cultivated  artificially. 

Trypanosomiasis  of  South  America  is  not  unlike  sleeping-sick- 
ness of  Africa.  It  is  caused  by  Tr.  cruzi,  a  parasite  of  eight  spores 
developing  in  organs,  serum  or  red  cells.  It  is  transmitted  by 
Conorrhinus  megistus,  a  large  insect. 

In  Dum  Dum  fever  or  Kala  Azar,  a  disease  characterized  by 
wasting,  anemia,  fever  and  splenomegaly  occurring  in  India, 
curious  bodies,  called  Leishmann-Donovan  bodies,  have  been 
found.  These  resemble  the  malarial  plasmodia  roughly,  and  if 
cultivated  on  blood  agar  elongated  herpetomas-like  bodies  with- 
out undulating  membranes  will  develop.  They  are  to  be  found 
in  the  juice  obtained  by  splenic  puncture  lying  within  cells,  espe- 
cially endothelium  and  large  lymphocytes;  on  rare  occa- 
sions they  have  been  met  in  the  blood.  The  transmission  is 
not  certainly  known  but  may  be  by  the  bed-bug  or  by  fleas. 

Trichomonas  vaginalis  and  intestinalis.  are  flagellates  which 
are  apparently  able  to  set  up  some  inflammatory  irritation  in  the 
places  indicated  by  their  special  names. 

TREPONEMA  PALLIDUM  (Schaudinn) 

(Spirochaeta  Pallida.) 

Treponema  Pallidum.— There  has  been  some  discussion  as  to 
the  proper  classification  but  now  this  organism  is  usually  placed 
among  the  Flagellata,  genus  Treponema.  It  does  not  possess  an 
undulating  membrane,  is  flagellated,  is  of  stiff  and  regular  shape, 
and  multiplies  by  longitudinal  division. 

Morphology  and  Stains. — This  organism  is  extremely  delicate 
in  structure,  from  4  to  14/4  in  length  and  about  .3/4  in  width;  has 


TREPONEMA  PALLIDUM  241 

from  3  to  12  turns  or  bends,  and  its  ends  are  delicately  pointed. 
Its  curves  form  a  large  arc  of  a  small  circle;  the  Sp.  refringens 
curves  form  a  small  arc,  frequently  irregular,  of  a  larger  circle. 
It  multiplies  by  both  transverse  and  longitudinal  division.  As 
this  organism  is  stained  with  difficulty  it  requires  a  special  one, 
that  of  Giemsa  yielding  the  best  results.  Aniline  gentian  violet, 
Romanowsky's,  and  Leishman's  stains  also  color  it.  It  may  be 
stained  in  tissues  by  silver  and  pyrogallic  acid  methods. 

Habitat. — It  has  not  been  found  in  tissues  of  normal  persons,  or 
those  ill  with  carcinoma,  tuberculosis,  etc.,  but  only  in  the  tissues 
of  individuals  suffering  with  syphilis.  It  is  a  strict  parasite. 

Vitality. — The  organism  is  readily  destroyed  by  the  ordinary 
disinfectants  and  dies,  after  a  few  minutes'  exposure  to  5o°C. 

The  Treponema  pallidum  has  now  fulfilled  the  postulates  of 
Koch.  It  can  be  cultivated  from  human  lesions  (with  some 
difficulty  to  be  sure),  it  can  be  implanted  in  animals  (monkeys 
and  rabbits)  and  there  reproduce  syphilitic  lesions;  and  it  can  be 
re-cultivated  from  them.  In  these  experimental  diseases  it  re- 
tains the  proper  morphology.  According  to  Noguchi  there  are 
two  types,  a  slender  and  a  stout,  which  breed  true  to  these  charac- 
ters and  correspond  to  slight  pathogenic  variations.  Noguchi 
succeeded  in  cultivating  the  Tr.  pall,  in  pure  culture  by  using 
the  juice  from  human  or  monkey's  lesions  or  from  the  syphilitic 
orchitis  of  rabbits.  This  he  grows  in  serum  water  or  serum  agar 
to  which  has  been  added  fresh  tissue  of  rabbit.  The  organism 
grows  as  fine  fibrils  in  arborescent  colonies.  These  can  be  selected 
pure  by  cutting  the  tube  and  the  agar  column.  Motion  is  of 
screw  and  serpentine  character.  No  odor  or  spores  are  produced. 
This  organism  must  be  imagined  and  remembered  as  a  corkscrew 
and  not  a  waving  line.  The  Gram  stain  is  negative. 

From  the  cultures  of  this  organism  a  toxic  extract  can  be  ob- 
tained which,  when  rubbed  into  the  skin  of  a  syphilitic  in  the 
late  stages,  gives  a  typical  skin  reaction,  luetin  and  the  luetin 
reaction. 

16 


242  ANIMAL   PARASITES 

The  Spiroch&ta  refringens,  which  has  been  also  cultivated  by 
Noguchi  and  thought  by  him  to  be  a  Treponema  also,  grows  with- 
out fresh  animal  tissue  in  a  short  time  and  produces  no  odor. 

Pathogenesis. — It  has  been  found  in  chancre,  condylomata,  and 
mucous  patches  in  the  early  stages  of  syphilis;  also  in  the  blood, 
blister-fluids,  spleen,  bone  marrow,  liver,  thymus  gland,  and 


FIG.  76. — The  Spirochaeta  refringens  is  the  larger  and  more  darkly  stained 
organism,  while  the  lightly  stained  and  more  delicate  parasite  is  the  Spiro- 
chaeta pallida  (Treponema  pallidum).  From  a  chancre  stained  with  Wright's 
blood  stain.  (Hirsch — by  Rosenberger.) 

lymphatic  glands,  and  in  the  brain  and  cord  of  taboparetics. 
Associated  with  this  organism,  in  nearly  every  case,  is  a  coarse- 
looking  larger  spirochaeta  (Treponema),  which  stains  deeper,  and 
has  been  called  the  Spirochaeta  (Treponema)  refringens  (q.v.). 
In  a  series  of  experiments,  Metchnikoff  and  Roux  caused  abor- 
tion of  the  chancre  following  inoculation  of  syphilitic  virus  on  the 
eyelid  of  a  chimpanzee,  by  calomel  inunction  carried  out  less  than 
one  hour  after  the  infection;  a  solution  of  sublimate  has  not  the 
same  prophylactic  property. 


RELAPSING   FEVER   ORGANISM  243 

It  does  not  require  any  intermediate  host  for  transmission  as 
do  the  recognized  animal  parasites  of  malaria  and  filariasis,  etc. 

Treponema  pertenue  is  the  organismal  cause  of  Frambcesia 
or  Yaws,  a  cutaneous  and  general  infection  of  the  tropics  similar 
to  syphilis.  It  is  about  the  size  and  shape  of  the  syphilis  spiro- 
chete  but  is  distributed  differently  in  the  human  lesions,  being 
more  in  the  outer  skin  and  less  in  the  vicinity  of  blood-vessels. 
It  has  not  been  cultivated. 

Spirochaeta  nodosa  (Huebner)  or  icterohemorrhagica  (Inada) 
is  believed  to  be  the  cause  of  Weil's  disease.  The  spiral,  a  tiny 
organism  about  5  micra  long  is  found  in  the  blood,  liver  and 
kidneys.  It  is  transmissible  to  guinea  pigs.  Rats  are  supposed 
to  be  the  means  of  transfer  since  several  varieties  probably 
harbor  the  parasite.  Excretion  of  the  organism  takes  place  via 
the  kidneys.  Spirocheticidal  substances  occur  in  the  blood  in  a 
favorably  progressing  attack. 

Spirochseta  morsus  muris  and  Sp.  muris  ratti  are  supposed 
to  be  the  cause  of  rat  bite  fever.  These  organisms,  resembling 
but  grosser  than  the  Treponema  pallidum,  enter  with  a  rat  bite 
and  can  be  found  in  swollen  drainage  lymph  nodes. 

RELAPSING  FEVER  ORGANISM 

European  Relapsing  Fever. — Caused  by  Treponema  of  Spiro- 
chseta obermeieri. 

African  Relapsing  Fever. — Caused  by  Trep.  or  Sp.  duttoni, 
transmitted  by  tick  Ornithodorus  moubata. 

American  Relapsing  Fever. — Caused  by  Trep.  or  Sp.  Novii. 

Bombay  Relapsing  Fever. — Caused  by  Trep.  or  Sp.  carteri. 
The  transmission  of  the  first,  third  and  fourth,  while  not  definitely 
known,  is  probably  by  a  louse;  ticks  may  also  be  responsible. 

Morphology. — They  have  lately  been  cultivated  and  retain 
somewhat  of  their  virulence  for  monkeys  and  rodents.  They  are 
elongated,  flexible,  corkscrew-like,  serpentine  and  vibratory  in 


244  ANIMAL  PARASITES 

motility,  and  do  not  form  spores.  There  is  a  single  terminal 
flagellum.  They  are  stained  with  reasonable  ease  by  plychrome 
methods,  especially  Giemsa,  but  not  by  Gram's  method.  They 
measure  from  10  to  40/1  in  length  and  about  i/*  in  breadth. 
Coils  vary  from  6  to  20.  The  American  type  is  smaller  than  the 
rest. 


FIG.  77. — Spirilla  of  relapsing  fever  from  blood  of  a  man.     (Kolle  and 

Wassermann.) 


Transmission. — The  known  tick  which  transmits  these  organ- 
isms becomes  infective  in  one  week  after  biting  a  patient  and 
remains  so  all  its  life;  its  young  are  also  infective.  The  types  of 
disease  vary  but  little.  In  all  these  is  a  relasping  fever  with 
periods  of  apyrexia  in  between.  During  the  fever  the  spirochsetes 
are  swimming  free  in  the  blood  and  disappear  in  the  afebrile 
interval. 

Cultivation. — They  are  cultivated  in  the  manner  given  for 
Trep.  pallidum  by  Noguchi,  by  adding  citrated,  therefore  de- 
fibrinated,  blood  to  serum  or  ascitic-fluid-fresh-tissue-agar.  They 
breed  true  to  type.  They  remain  alive  several  days  under 
favorable  artificial  conditions  but  cannot  be  cultivated  after 


SPOROZOA  245 

they  have  left  the  body  a  few  hours  without  being  on  suitable 
culture  media. 

The  periods  of  fever  last  from  five  to  seven  days,  when  a  crisis 
occurs.  After  an  apyrexial  period  the  fever  recurs.  The  spiro- 
chaetae  are  found  in  great  numbers  in  every  microscopical  field. 

In  the  apyrexial  period  the  spleen  becomes  engorged  and  the 
leucocytes  devour  the  parasites.  Monkeys  with  excised  spleens 
are  more  susceptible  to  infection  than  others. 

Immunity. — The  blood  from  rats  that  have  been  immunized 
by  repeated  injections  of  blood  from  spirochetal  rats,  if  injected 
into  other  rats,  is  capable  of  conferring  an  immunity  on  them  by 
causing  spirochaetes  to  disappear  from  their  blood.  One  attack 
seems  to  confer  immunity  to  the  special  form  causing  it  but 
probably  not  to  the  others. 

SPOROZOA 

The  most  important  of  this  family  are  the  malarial  parasites 
(which  belong  to  the  order  Haemosporidia),  and  the  Coccidia. 

In  general  the  sporozoa  are  unicellular  organisms  that  lead  a 
parasite  existence  in  the  tissues,  especially  cells,  of  higher  ani- 
mals. They  ingest  liquid  food,  have  no  cilia  in  the  adult  stage, 
and  flagella  are  possessed  only  by  the  males.  There  may  be  one 
or  more  nuclei.  Propagation  is  effected  by  spores,  but  budding 
and  division  do  occur,  though  rarely.  Alternate  generation 
takes  place  frequently. 

MALARIAL  PARASITES 

Haemosporidia  of  Man. — The  most  important  disease  caused 
in  human  beings  by  the  haemosporidia  is  malaria,  or  ague,  and 
excepting  the  deserts,  mountains,  and  arctic  regions,  this  disease 
is  very  widely  distributed. 

Three  different  parasites  producing  different  clinical  entities 
are  known.  According  to  the  time,  frequency,  and  order  of  the 


246  ANIMAL   PARASITES 

outbreak  of  chills  and  fever,  various  clinical  names  have  been 
given  to  the  manifestation  of  the  disease.  Mannaberg  has 
arranged  the  following  scheme  to  show  the  different  forms  of 
outbreaks.  The  numbers  apply  to  the  paroxysms.  Each 
developmental  cycle  is  numbered  alike: 

i  i  i  i  i  i  i.  Simple  quotidian  fever. 

I  o  i  o  i  o  i  Simple  tertian  fever. 

looiooiooi.  Simple  quartan  fever. 

12121212.  Double  tertian  fever.  (Two  infections.) 

.123123123.  Triple  quartan  fever.          (Three  infections.) 

120120120.  Double  quartan  fever.          (Two  infections.) 

The  figures  refer  to  days  on  which  paroxysms  of  fever  occur. 
The  o  represents  the  afebrile  day. 

PLASMODIUM  MALARIA  (Laveran) 

This  is  the  quartan  parasite,  and  produces  in  man,  in  cases  of 
one  infection,  paroxysms  of  fever  every  fourth  day. 

It  appears  in  the  blood,  after  a  paroxysm,  as  a  small  non-pig- 
mented  body  on  the  bodies  of  the  red  blood  cells.  It  has  feeble 
amoeboid  motion;  slowly  penetrates  the  corpuscle,  and  specks  of 
melanin  appear  in  its  protoplasm.  Forty-eight  hours  after  the 
attack  the  parasite  measures  from  one-half  to  two-thirds  the 
size  of  the  red  cell.  Sixty  hours  after  the  paroxysm — twelve 
before  the  next — the  parasite  completely  fills  the  red  cell,  leav- 
ing xonly  a  narrow  rim,  which  later  on  disappears.  Six  hours 
before  the  next  paroxysm,  shizogony  begins.  The  grains  of 
melanin  are  arranged  like  the  spokes  of  a  wheel,  and  then,  leaving 
the  radii,  crowd  above  the  centre  (the  rest  of  the  cell  being  pig- 
mentless)  gradually  dividing  into  8  or  12  pear-shaped  bodies,  or 
merozoites.  These  separate  from  each  other  and  individually 
attack  a  fresh  red  cell,  and  this  attack  brings  another  paroxysm 
of  fever  seventy-two  hours  after  the  previous  one.  The  grains 


PLASMODIUM  VIVAX  247 

of  pigment  are  taken  up  by  the  leucocytes  and  deposited  in  the 
spleen  and  bone  marrow. 

The  nucleus  of  the  parasite  may  be  seen  if  suitably  strained. 
The  double  or  triple  quartan  is  explained  by  the  fact  that  there 
are  two  or  three  groups  of  organisms  that  undergo  sporogony  at 
periods  separated  from  each  by  twenty-four  hours. 

PLASMODIUM  VIVAX  (Grassi) 

The  cause  of  tertian  fever  occurring  in  the  spring.  It  differs 
from  the  Plasmodium  malaria  because  of  shorter  period  (forty- 
eight  hours)  consumed  in  schizogony  (or  sporulation),  the  much 
greater  activity  of  the  amoeboid  movement,  and  the  affected  cor- 
puscles becoming  enlarged;  also  by  the  fact  that  many  of  the 
melanin-bearing  stages  are  visible.  The  shizogony  is  rarely 
apparent  in  the  circulating  blood,  but  in  the  spleen  these  stages 
are  easily  seen.  There  are  from  15  to  20  merozoites  (segmented 
bodies  or  spores)  which  are  arranged  in  an  irregular  heap,  but  not 
radially  like  wheel  spokes.  The  merozoites  are  smaller  than  the 
quartan  variety  and  are  more  numero'us.  The  flagellated  form 
can  but  rarely  be  seen  in  the  freshly  drawn  blood.  If  some  blood, 
containing  the  large  extracorpuscular  bodies,  is  put  in  a  moist 
chamber,  they  throw  out  flagella.  These  flagella  are  really 
micro  gametes  and  are  sexually  active.  The  extracorpuscular 
bodies  are  partly  macro gametes ,  and  if  they  become  flagellated 
they  are  called  polymites,  and  are  the  micro gametocytes.  The 
merozoites,  or  spores,  finally  burst  forth  from  the  erythrocytes, 
starting  again  another  cycle  (attended  with  a  paroxysm  of  fever) . 
These  spores  appear  in  the  freshly  invaded  corpuscles  as  hyaline 
bodies  with  slight  movement.  As  they  grow  in  size,  pigment 
appears  in  the  protoplasm.  Certain  of  these  do  not  break  up 
into  merozoites,  or  spores,  but  become  extracellular  bodies 
gametocytes  of  male  or  female  character.  There  may  be  two 
infections  in  which  schizogony  occurs  every  other  day  in  alternate 
days,  12121212. 


248  ANIMAL   PARASITES 

PLASMODIUM  FALCIPARUM 

The  plasmodium  of  aestivo-autumnal  fever,  or  pernicious  ma- 
larial fever,  also  called  tropical.  The  outbreaks  of  this  occur  ir- 
regularly. The  disease  produced  by  them  is  very  much  more 
malignant  and  is  harder  to  cure.  The  young  spore  appears  in 
the  corpuscle  as  a  small  hyaline  body,  smaller  than  the  other 
forms  and  much  more  active.  The  size  and  shape  of  the  red  cells 
are  little  if  any  altered  but  they  become  granular  and  polychro- 
matophilic.  The  pigment  is  very  finely  granular  and  the  body 
frequently  presents  the  signet-ring  appearance.  There  may  be 
more  than  one  parasite  to  a  red  cell.  The  cycle  of  development 
(schizogony)  is  twenty-four  to  forty-eight  hours.  The  plas- 
modium in  its  schizogony  divided  into  8  to  24  merozoites  or  spores, 
and  are  arranged  in  a  spore-like  form.  The  extracorpuscular  bod- 
ies may  resemble  a  crescent  or  sickle;  this  form  is  very  character- 
tic  of  aestivo-autumnal  fever.  There  are  two  forms  of  these 
crescents,  one  delicate,  the  male,  and  one  larger  and  ovoid,  the 
female.  They  are  very  resistant  to  quinine  and  persist  for  a  long 
period  in  the  blood.  Plasmodia  undergoing  schizogony  are  often 
found  in  the  brain  capillaries  after  death,  which  accounts  for  the 
cerebral  symptoms  in  such  cases.  This  form  can  be  differentiated 
from  the  others  by  the  irregular  and  pernicious  type  of  fever  pro- 
duced; by  its  great  resistance  to  quinine;  the  fewer  number  of 
merozoites;  the  finely  granular  appearance  of  the  pigment;  the 
relatively  small  size  of  the  young  intracorpuscular  body;  and, 
by  the  ring  shape  of  some  of  the  young  forms. 

Often,  in  blood  from  malarial  cases,  pigmented  leucocytes  are 
seen,  and  ghost,  or  shadow,  red  corpuscles  from  which  the  haemo- 
globin has  been  dissolved  are  often  met  with.  Spherical  extra- 
corpuscular  bodies  become  flagellated  (gametes)  in  freshly  drawn 
blood.  The  parasite  may  be  studied  in  fresh  film  preparations 
and  by  staining  dried  films  by  methylene  blue  and  eosin,  Roma- 
nowsky's,  or  Jenner's  methods.  They  are  much  more  frequent 
in  the  pyrexial  period,  and  when  quinine  has  not  been  given. 


PLASMODIUM   FALCIPARUM  249 

The  various  plasmodia  are  transmitted  to  man  invariably  by 
the  anopheles  mosquito,  in  the  bodies  of  which  they  undergo  a 
different  (sexual)  existence.  It  has  been  positively  demonstrated 
that  the  various  plasmodia  undergo  an  alteration  of  generations 
and  require  two  different  hosts  for  their  development,  i.e.,  mos- 
quito, man. 

The  asexual  development,  or  schizogony,  takes  place  in  the 
blood  of  man,  the  sporogony,  or  sexual  development,  in  the  body 
of  the  anopheles  mosquitoes,  the  bite  of  which  sets  up  an  infec- 
tion in  man,  since  the  sporozoites  of  the  various  plasmodia  are 
developed  in  the  salivary  glands  of  these  mosquitoes.  In  the 
act  of  biting,  the  sporozoites  reach  the  erythrocytes  where  they 
become  the  intracorpuscular  hyaline  bodies  beginning  again  their 
asexual  cycle  of  development  in  the  blood. 

That  the  mosquito  is  the  intermediate  host  of  the  malarial  para- 
site and  that  the  infection  in  man  follows  bites  by  infected  mos- 
quitoes has  been  abundantly  proven.  The  mosquitoes  that  act 
in  this  way  are  the  various  Anopheles;  the  Anopheles  maculipennis 
being  the  offender  most  frequently.  The  freshly  formed  schizonts 
in  the  blood  of  an  infected  man  are  conveyed  into  the  intestines 
of  the  mosquito.  Here  sexual  reproduction  of  the  parasite  begins. 
The  male  elements,  flagellar  microgametes  penetrate  the  female 
elements,  macrogametes  (cellular),  and  after  a  time  there  appear 
intra-cellular  fusiform  bodies,  ookinets.  These  bore  into  the 
intestinal  walls  of  the  mosquito  and  there  remain.  After  a  time 
they  are  converted  into  round  bodies,  or  ob'cysts.  The  nucleus 
of  the  oocysts  divides  rapidly  and  other  daughter  nuclei  are  formed 
and  new  cells  called  sporoblasts.  After  about  eight  days  these 
form  the  sporozoites.  The  number  of  sporozoites  in  each  oocyst 
varies  from  hundreds  to  many  thousands  (often  10,000).  These 
oocysts  burst  and  the-  sporozoites  in  the  circulation  find  their 
way  to  the  salivary  glands  of  the  mosquito.  When  a  mosquito 
bites  a  human  being  they  are  introduced  into  the  blood  where 
they  are  quickly  transformed  into  the  intracellular  hyaline  bodies 


DESCRIPTION  OF  FIG.  78 

Life  history  of  malaria  parasite,  Plasmodium.  i,  Sporozoite,  introduced 
by  mosquito  into  human  blood,  the  sporozoite  becomes  a  schizont;  2,  young 
schizont;  3,  young  schizont  in  a  red  blood  corpuscle;  4,  full-grown  schizont; 
5,  nuclear  division;  6,  spores,  or  merozoites,  from  a  single  mother-cell;  7,  young 
macrogamete  (female),  from  a  merozoite,  and  situated  in  a  red  blood  cor- 
puscle; 7a,  young  microgametoblast  (male);  8,  full-grown  macrogamete;  8a, 
full-grown  microgametoblast;  9,  mature  macrogamete;  ga,  mature  micro- 
gametoblast; 96,  resting  cell,  bearing  six  flagellate  microgametes  (male); 
10,  fertilization  of  a  macrogamete  by  a  motile  microgamete;  the  macrogamete 
next  becomes  an  ookinete;  n,  ookinete,  or  wandering  cell,  which  penetrates 
into  the  wall  of  the  stomach  of  the  mosquito;  12,  ookinete  in  the  outer  region 
of  the  wall  of  the  stomach,  i.e.,  next  to  the  body  cavity;  13,  young  oocyst, 
derived  from  the  ookinete;  14,  oocyst,  containing  sporoblasts,  which  develop 
into  sporozoites;  15,  older  oocyst;  16,  mature  oocysts,  containing  sporozoites; 
17,  transverse  section  of  salivary  gland  of  an  Anopheles  mosquito,  showing 
sporozoites  of  the  malaria  parasite  in  the  gland  cells  surrounding  the  central 
canal. 

1-6  illustrate  schizogony  (asexual  production  of  spores);  7-16,  sporogony 
(sexual  production  of  spores). 

(FOLSOM — After  GRASST  and  LEUCKART,  by  permission  of  Dr.  Carl  Chun.) 


MALARIAL  PARASITES 


16 


FIG.  78. 


252 


ANIMAL   PARASITES 


and  begin  their  asexual  sporogony  in  the  blood.  Each  develop- 
mental cycle  causing  a  febrile  paroxysm  either  every  day  or  alter- 
nate days,  or  on  every  fourth  day,  etc.,  depending  on  the  character 
of  the  organisms  and  the  number  of  infections.  To  prevent 


FIG.  79. — Coccidium  ho  minis,  from  intestine  of  rabbit:  i,  a  degenerate  epi- 
thelial cell  containing  two*coccidia;  2,  free  coccidium  from  intestinal  contents; 
3,  coccidium  with  four  spores  and  residual  substances;  4,  an  isolated  spore;  5, 
spore  showing  the  two  falciform  bodies — X  1140.  (From  Railliet,  in  Tyson's 
Practice.) 

spread  of  malaria,  mosquitoes  must  be  prevented  from  reaching 
individuals  infected  with  malaria  and  those  not  infected.  Screens 
accomplish  this  best.  The  larva  of  the  mosquito  develops  in 
stagnant  water.  To  prevent  the  development  of  these  young 
mosquitoes  oil  should  be  poured  on  the  water,  thus  cutting  off 
the  air  and  means  of  respiration. 


COCCIDIUM  253 

Bass,  of  New  Orleans,  claims  to  have  successfully  cultivated 
malarial  plasmodia  of  the  species  vivax  and  falciparum  by  the  use 
of  human  blood.  He  has  also  succeeded  when  using  Locke's  fluid 
minus  calcium  chloride  plus  ascitic  fluid.  One-half  percent  dex- 
trose is  usually  added.  The  blood  is  drawn,  so  that  it  can  be 
defibrinated,  into  small  flat-bottom  tubes.  These  are  incubated 
at  4o°C.  The  column  of  fluids  is  1-2  inches  high,  the  clear  serum 
layer  being  %  inch  at  least.  The  parasites  grow  in  the  upper 
layer  of  the  cellular  sediment.  Undiluted  serum  and  leucocytes 
are  lytic  for  plasmodia.  For  renewed  cultures  these  must  be 
removed  but  uninjured  red  cells  must  be  added.  Only  the  asexual 
division  has  been  observed.  Leucocytes  phagocyte  pll  free 
parasites  under  artificial  conditions. 

COCCIDIUM 

Coccidium  hominis  is  another  member  of  the  sporozoa  that  occa- 
sionally infects  man.  Coccidia  are  infectious  also  for  horses, 
goats,  oxen,  sheep,  pigs,  guinea  pigs,  weasels  and  rabbits.  The 
organism  is  essentially  a  cell  parasite  inhabiting  the  cells  of  the 
gastro-intestinal  tract  by  preference,  chiefly  the  liver  and  intes- 
tinal mucous  membranes.  They  lead  a  sexual  and  asexual 
existence  like  the  malarial  parasites  (alternate  generation).  The 
young  sickle-shaped  nucleated  sporozoite  penetrates  an  epithelial 
cell,  where  it  gradually  develops,  ultimately  dividing  into  numer- 
ous sporozoites.  This  is  the  asexual  stage  of  development 
(schizogony) ,  the  sexual  stage  being  called  sporogony. 

The  sporozoites  are  differentiated  into  the  two  sex  elements. 
These  are  large  granular  appearing  cells;  the  male  being  smaller, 
divides  into  numerous  flagellated  microgamates  that  penetrate  the 
female  granular  cells,  macrogametes,  and  fertilize  them.  These 
fertilized  macrogametes,  or  zygotes,  form  capsules  and  become 
oocysts  which  divide  into  numerous  sporoblasts,  changing  into 
sickle-shaped  sporozoites  upon  liberation. 


254 


ANIMAL  PARASITES 


The  coccidia  are  easily  demonstrable  in  tissue  and  in  faeces. 
They  produce  in  man  occasionally  a  fatal  disease  infecting  the 
liver  and  intestines.  Cattle  sometimes  die  from  haemorrhagic 


FIG.  80. — Development  of  coccidium  cuniculi:  a,  b,  c,  young  coccidia  in  epi- 
thelial cells  of  gall  duct;  d,  e,f,  fully  grown  encysted  coccidia;  g,  h,  i,  k,  /, 
showing  development  of  spores;  m,  isolated  spore,  greatly  magnified,  showing 
the  two  falciform  bodies  (pseudonawcdla;  sporozoites}  in  natural  position;  n,  a 
spore  compressed  so  as  to  separate  the  two  srjorozoites,  o,  a  sporozoite  or 
falciform  body  with  y,  its  nucleus.  (From  Railliet  after  Balbiani — in  Tyson's 
Practice.) 

dysentery  due  to  one  of  the  coccidia.  The  disease  is  transmitted 
by  the  ingestion  of  food  contaminated  by  faeces  containing  the 
sporozoites. 

Acid  f  uchsin  stains  the  sporozoa. 


CHAPTER  X 
THE  FILTERABLE  VIRUSES 

This  general  term  means  that  the  virus  of  a  disease  can  pass 
through  a  porcelain  filter  and  usually  that  it  cannot  be  seen  by  the 
microscope.  It,  however,  does  not  mean  that  it  is  invisible  at  all 
stages  since  in  one  case  at  least  we  have  been  able  by  means  of  the 
ultramicroscope  to  see  what  is  almost  certainly  the  particular 
causal  agent.  Again  it  is  said  that  spirochaetes  when  young  will 
traverse  porcelain  niters.  The  term  will  cover  in  this  chapter 
those  diseases  of  importance  to  man  whose  causal  agents  cannot 
be  morphologically  described,  but  whose  characters  are  more  or  less 
well  known.  The  list  of  diseases  caused  by  submicroscopic  agents 
is  as  follows:  African  horse  sickness,  swamp  fever  of  horses,  catar- 
rhal  fever  of  sheep,  yellow  fever,  Dengue,  three-day  fever,  typhus 
fever,  poliomyelitis,  rabies,  variola,  with  its  congeners  vaccinia 
and  animal  pox,  hog  cholera,  foot  and  mouth  disease,  fowl  plague, 
fowl  diphtheria,  transplantable  sarcoma  and  leukemia  of  fowls, 
cattle  plague,  trachoma,  pleuropneumonia  of  cattle,  molluscum 
contagiosum,  measles,  scarlet  fever,  guinea-pigs  epizootic  and 
some  diseases  of  plants.  As  said  above,  only  the  diseases  trans- 
missible to  human  beings  are  reviewed. 

Some  of  the  above  diseases,  notably  rabies,  scarlatina  and  tra- 
choma, show  in  the  leucocytes  and  epidermal  cells  certain  struc- 
tures or  inclusion  bodies  to  which  the  name  Chlamydozoa  was  given 
by  Prowaczek,  a  term  implying  that  a  parasitic  body  is  growing  in 
a  mantle.  They  start  as  tiny  specks  in  the  cytoplasm  shortly 
found  to  be  surrounded  by  a  clear,  sharply  outlined  halo.  They 
seem  to  grow  at  the  expense  of  the  host  cell.  Their  exact  char- 
acter is  not  understood;  they  are  probably  evidences  of  cellular 
degeneration  under  the  influence  of  some  noxa. 

255 


256  THE   FILTERABLE  VIRUSES 

Hydrophobia. — This  disease  has  long  been  considered  to  be  an 
infectious  one,  but  the  causal  parasitic  agent  has  never  been  dis- 
covered. It  is  commonly  found  in  dogs,  cats,  wolves,  rabbits,  etc., 
but  other  domestic  animals,  and  man  may  become  infected.  It  is 
a  disease  of  the  central  nervous  system,  highly  infectious,  always 
following  a  bite  or  other  injury  in  which  the  skin  is  broken,  and 
in  which  lesion  the  virus  may  be  deposited.  Infection  may  be 
caused  by  injecting  emulsified  infected  nerve  tissue  (brain)  into 
susceptible  animals  (rabbits  or  monkeys).  The  disease  is  always 
fatal  after  it  is  well  established.  Well-marked  histological  lesions 
of  the  central  nerve  tissues,  particularly  the  large  ganglia,  have 
been  found  by  Van  Gehutchen  and  Nelis,  and  Ravenel  and  Mc- 
Carthy. If  emulsified  brain  tissue  from  an  animal  that  has  died  of 
hydrophobia  is  filtered  through  a  "germ-proof  "  filter  the  nitrate  is 
capable  of  setting  up  the  disease  in  a  healthy  animal  if  it  is  injected 
into  it.  By  long  centrifugation  of  emulsified  infected  brain  tissue, 
the  supernatant  fluid  loses  its  power  of  reproducing  the  disease  on 
injection.  Virus  may  also  be  found  in  mammary  and  lacrymal 
secretions,  pancreas,  cerebro-spinal  fluid  and  aqueous  humor. 

The  organism  is  toxic  in  character,  since  filtrates  sometimes  fail 
to  produce  transmissible  disease,  but  emaciation,  paralysis,  and 
death  are  caused  by  their  injection  into  rabbits,  the  tissues  of 
which,  in  turn,  are  not  infectious. 

The  unknown  organisms  are  rather  resistant  to  agents  that  are 
germicidal.  They  are  destroyed  in  fifty  minutes  by  a  5  percent 
carbolic  solution,  and  in  three  hours  by  a  1-1,000  corrosive  subli- 
mate solution.  Direct  sunlight  kills  them  quickly,  as  do  radium 
emanations.  The  latter  have  been  used -as  curative  measure 
with  reputed  success.  A  temperature  from  52°  to  58°C.  for  one- 
half  hour  destroys  them,  but  they  resist  extreme  cold  of  liquid  air 
(—312°)  for  many  weeks.  Pasteur  found  that  desiccation  attenu- 
ated the  virus.  Chlorine  kills  it  quickly,  while  glycerine  does  not. 
The  virus  may  be  increased  in  virulence  by  passing  the  "  street 
virus"  of  dogs  through  a  series  of  rabbits.  Here  the  period  of 


HYDROPHOBIA 


257 


incubation  decreases  from  three  weeks  to  six  days,  but  beyond 
this  the  period  does  not  become  less,  and  the  degree  of  virulence 
from  the  virus  lead  Pasteur  to  name  it  virus  fixe  (fixed  virus). 

Passing  the  virus  through  foxes,  cats,  and  wolves  also  intensifies 
the  virulence,  while  monkeys  and  chickens  attenuate  it. 


FIG.  81. — Section  through  the  cornu  ammonis  of  brain  of  a  rabid  dog; 
lined  by  the  method  of  Lentz.  Five  Negri  bodies  of  different  sizes  are 
>wn,  enclosed  within  the  ganglion  cells.  The  smallest  contains  only  three 
tiute  granules.  (After  Lentz,  Centralbl  f.  Bakt.,  1907,  Abt.  I,  Vol.XLIV, 
>•  378.) 

Negri  bodies,  intracellular  bodies  discovered  by  Negri,  are 
>und  in  the  ganglionic  cells  of  rabid  animals.  These  bodies  stain 
>y  eosin,  and  are  from  i  to  27;*  in  size,  being  generally  about  s/x. 


258  THE   FILTERABLE   VIRUSES 

They  are  found  particularly  in  the  cornu  of  Ammon;  in  Purkinje's 
cells  in  the  cerebellum;  and  in  the  larger  cells  of  the  cortex  of  the 
cerebrum.  These  may  be  the  cause  of  the  disease,  but  there  are 
several  objections  to  this  hypothesis.  Their  distribution  does  not 
correspond  to  the  parts  of  the  nervous  system  that  are  most 
intensely  affected  in  hydrophobia,  i.e.,  medulla  and  pons.  In  the 
latter  locality  these  bodies  are  rarely  encountered.  They  are  not 
found  invariably  in  animals  dead  from  rabies,  and  are  considered 
to  be  too  large  to  pass  through  a  Berkefeld  filter;  this  latter  view 
may  not  be  a  correct  one.  The  finding  of  these  bodies  has  been 
considered  by  Negri  to  be  good  grounds  for  considering  the  case 
to  be  hydrophobia.  The  rapid  diagnosis  of  the  disease  in  animals 
can  only  be  effected  by  killing  them  and  examining  the  nervous 
tissues,  or  inoculating  other  animals  with  them.  Histologically, 
three  marked  changes  may  be  noted:  (i)  The  finding  of  the  Negri 
bodies.  (2)  The  finding  of  the  degeneration  of  the  cells  of  the 
larger  ganglia  with  the  proliferation  of  the  endothelial  cells  lining 
the  ganglionic  spaces  (Van  Gehutchen  and  Nelis).  (3)  The  find- 
ing of  certain  tubercles  in  the  medulla,  which  are  called  Babes 
tubercles,  though  these  are  not  wholly  characteristic,  as  they  are 
found  in  other  diseases.  Hydrophobia  is  transmitted  from  the 
site  of  the  wound  to  the  central  nervous  tissues  by  the  nerves,  and 
the  incubation  period  varies  with  the  distance  of  the  wound  from 
the  central  nervous  system;  the  majority  of  cases  occur  between 
twenty  to  sixty  days  after  a  bite. 

Immunity  against  infection  and  the  development  of  the  disease 
after  the  reception  of  an  infected  wound,  may  be  accomplished  by 
Pasteur's  method  (see  chapter  on  Vaccine). 

Yellow  Fever 

That  this  disease  is  caused  by  a  parasite  there  can  be  no  doubt. 
It  is  highly  infectious  and  largely  confined  to  the  tropical  regions' 
of  the  western  hemisphere  and  in  parts  of  Africa.  Resembling 
diseases  established  as  due  to  protozoa,  this  one  is  unquestion- 


YELLOW   FEVER  2 59 

ably  spread  by  mosquitoes,  and  it  has  been  definitely  determined 
by  Carrol  and  Reed  that  the  female  Aedes  capolus  (formerly 
called  Stegomyia  fasciata  and  St.  calopus)  is  the  means  of  its 
propagation.  Carrol  believes  that  the  undiscovered  parasite  of 
yellow  fever  is  of  the  animal  kingdom,  for  the  following  reasons: 
(i)  It  is  absolutely  necessary  for  its  continued  existence  that  it 
undergoes  alternate  generation  in  man  and  in  the  Stegomyia 
mosquito.  This  is  peculiar  to  the  sporozoa.  (2)  The  fact  that 
twelve  days  must  elapse  before  the  mosquito  is  capable  of  infect- 
ing man  is  evidence  that  a  cycle  of  development  of  the  unknown 
parasite  is  taking  place  in  the  mosquito.  (3)  The  limitation  of  the 
cycle  of  development  of  the  parasites  to  a  single  genus  of  the 
mosquito  and  to  a  single  vertebrate  (man)  conforms  to  a  natural 
zoologic  law,  and  this  does  not  conform  to  our  knowledge  of  the 
life  history  of  bacteria.  (4)  The  effects  of  climate  and  tempera- 
ture on  the  life  history  of  the  Stegomyia,  and  on  the  rate  of 
development  of  the  parasites  in  the  bodies  of  the  mosquitoes  are 
exactly  the  same  as  the  effects  of  the  same  conditions  on 
the  anopheles  mosquito  and  the  malarial  parasite.  Without  the 
Stegomyia  there  can  be  no  yellow  fever.  Infection  requires  the 
fulfilling  of  the  following  conditions:  (i)  By  the  bite  of  the  mos- 
quito providing  the  insect  has  fed  on  the  blood  of  a  yellow  fever 
patient  within  the  first  three  days  of  the  fever.  (2)  The  disease 
is  not  transferred  immediately,  but  a  definite  incubative  period 
of  more  than  eleven  days  must  elapse  before  the  mosquito  can 
transfer  the  disease.  After  twelve  days  the  mosquito  has  been 
found  to  be  infected  for  at  least  fifty-seven  days.  (3)  Yellow 
fever  cannot  be  carried  by  fomites.  (4)  Yellow  fever  may  be 
produced  in  a  healthy  man  by  the  subcutaneous  injection  of  blood 
from  a  yellow  fever  case  (parasites  in  the  blood).  (5)  The  serum 
of  a  yellow  fever  patient  filtered  through  a  very  fine  Berkef eld  or 
porcelain  filter  is  still  capable  of  setting  up  the  disease  if  injected, 
proving  that  the  infection  agent  is  capable  at  some  stage  of  its 
life  to  pass  through  filter  pores.  (6)  An  attack  of  yellow  fever 


260  THE  FILTERABLE  VIRUSES 

produced  by  the  bite  of  a  mosquito  confers  immunity  against 
subsequent  infection.  (7)  The  period  of  infection  is  usually 
three  days  but  may  be  from  two  to  six  days.  (8)  A  house  or  ship 
may  be  said  to  be  infected  with  yellow  fever  only  when  there  are 
present  mosquitoes  capable  of  conveying  the  parasite  of  the 
disease.  (9)  The  spread  of  yellow  fever  may  be  prevented  by 
destroying  the  aedes  and  preventing  egress  and  ingress  of  the 
insects  from  yellow  fever  patients  to  the  non-immune.  (10)  No 
insect,  other  than  the  aedes,  has  been  found  to  be  concerned  in 
the  spread  of  yellow  fever. 

Noguchi  reports  the  discovery  of  a  tiny  spirochaete,  4-9/4  long 
and  .2;u  wide,  in  the  blood  of  yellow  fever  patients.  The  organism 
may  be  transferred  to  guinea  pigs  in  which  it  produces  lesions 
comparable  to  those  of  human  yellow  fever.  It  may  be  cultivated 
in  anaerobic  serum  water  tubes  and  it  will  pass  through  a  filter. 
During  an  attack,  either  human  or  artificial  in  a  guinea  pig, 
protective  anti-bodies  are  formed.  Spirals  are  only  found  in  the 
blood  in  the  first  few  days  of  an  attack.  They  do  not  stain  well 
and  can  best  be  seen  by  aid  of  the  dark  field  microscope.  It  is 
claimed  that  vaccination  with  these  spirals  produces  some  resist- 
ance to  yellow  fever. 

Yellow  fever  is  a  tropical  or  subtropical  disease,  because  the 
aedes  is  confined  to  these  regions,  and  the  disease  is  found 
in  low  moist  localities  rather  than  those  that  are  drier  and  higher, 
from  the  fact  that  the  mosquito  inhabits  the  former  and  not  the 
latter.  Yellow  fever  dies  out  after  the  first  sharp  frost,  because 
the, aedes  are  then  either  killed  or  undergo  hibernation.  Many 
conclusive  experiments  by  Reed  and  Carrol,  by  Guiteras,  and 
by  the  French  Commission  have  proved  that  the  aedes  is  beyond 
doubt  the  cause  of  the  spread  of  the  disease.  No_immunity,  other 
than  the  activity  acquired  one,  is  known. 

Small-pox  and  Vaccinia. — These  two  diseases  must  be  consid- 
ered to  be  but  two  clinical  activities  of  one  unknown  specific 
microorganism. 


SMALL   POX  26l 

Certain  protozoonoid  bodies  have  been  seen  by  numerous  ob- 
servers, notably  by  VanderLoeff,  L.  Peiffer,  and  Guarnieri.  The 
latter  gave  the  name  Cytoryctes  vaccinias  s.  variolas.  In  the 
deep  layers  of  the  epithelial  cells  of  the  pustules  of  vaccinia  and 
small-pox,  in  the  experimental  lesions  on  the  corneae  of  rabbits, 
and  in  the  protoplasm  of  the  cells,  these  bodies  are  found.  They 
are  about  the  size  of  a  micrococcus  and  exhibit  amoeboid  move- 
ments in  hanging-drop  preparations.  They  are  perfectly  char- 
acteristic of  the  lesion  produced  in  vaccinia  and  are  not  found  in 
other  diseased  conditions. 

In  variola  many  different  changes  occur  in  the  appearances  of 
these  cytoryctes,  suggesting  developmental  cycles. 

In  variola  they  are  often  intranuclear,  while  in  vaccine  they  are 
never  found  within  the  nuclei. 

The  cycle  of  development  is  suggestive  of  the  development 
of  many  of  the  protozoa.  Stages  of  development  exhibiting 
fusiform  amoeboid  shapes  can  be  seen,  and  pseudopodia  can  be 
detected  in  the  process  of  developmental  stages  suggestive  of 
gametocytes;  the  union  of  the  gametes  and  the  ultimate  forma- 
tion of  the  zygote  can  also  be  discerned. 

After  the  tenth  day  these  bodies  cannot  be  very  well  discerned 
in  the  tissues. 

There  is  reason  to  think  that  the  parasites  circulate  in  the  blood 
in  variola.  The  contagion  in  variola  is  thought  to  be  by  inhala- 
tion. It  is  certain  that  the  disease  can  be  produced  by  inocula- 
tion with  virus  from  a  case  of  small-pox.  The  contagion  exists 
in  the  scales,  pus  cells,  and  excretions  of  patients  ill  with  small-pox. 

If  the  virus  of  small-pox  is  introduced  into  a  monkey,  and  then 
into  a  cow  the  disease  produced  is  not  variola,  but  vaccinia 
(Monkman).  The  hypothetical  organism  above  described,  cyto- 
ryctes, becomes  attenuated  in  the  cow,  so  that  it  is  incapable  of 
producing  variola,  but  vaccinia. 

Rabbits,  horses,  and  sheep  are  susceptible  of  inoculation  with 
the  virus  of  vaccinia  (see  Vaccination).  Virus  may  be  tested  by 


262  THE  FILTERABLE  VIRUSES 

rubbing  over  the  shaven  bellies  of  rabbits,  setting  up  minute 
vesicles  and  finally  crusts  (Calmette). 

The  two  viruses,  that  of  variola  and  that  of  vaccinia,  are  now 
thought  to  be  identical.  In  a  diluted  condition  it  is  filterable. 
It  resists  drying  for  weeks  and  glycerine  eight  to  ten  months.  It 
is  destroyed  at  S7°C.  in  fifteen  minutes  and  easily  by  most  dis- 
infectants. Passive  immunization  has  not  been  achieved.  No- 
guchi  has  succeeded  in  growing  the  virus  in  the  testes  of  rabbits; 
one  of  his  objects  in  so  doing  was  to  afford  a  sterile  cultivation  of 
virus  which  might  be  used  for  preparing  vaccine. 

Scarlet  Fever 

Mallory  in  1903  found  certain  bodies  in  the  skin  of  scarlet  fever 
cases.  These  bodies,  he  assumed,  were  protozoan  in  character 
and  were  the  etiological  cause  of  the  disease.  He  named  them 
Cyclasterion  Scarlatinale.  They  have  been  found  rather  con- 
stantly in  the  skin  of  scarlet  fever  cases,  also  in  the  skin  in  cases 
of  measles  and  in  anti-toxin  rashes.  Dohle  has  also  described 
an  inclusion  body  within  the  polynuclear  leucocytes.  By  several 
observers  these  bodies  have  been  considered  to  be  artefacts  or 
degeneration  products  in  the  epithelial  cells. 

The  virus  of  scarlatina  is  now  considered  to  be  filterable  and 
transmissible  to  monkeys.  Mallory  has  lately  found  diphtheroid 
bacilli  in  great  numbers  in  the  pharynx  in  scarlatina  and  believes 
they  may  be  its  cause.  Virulent  streptococci,  usually  of  hemoly- 
tic  quality,  are  so  frequently  encountered  in  the  respiratory  tract 
and  in  complications  during  scarlet  fever  that  many  persons  look 
upon  them  as  the  cause;  this  cannot  be  proven  as  the  disease 
cannot  be  successfully  transmitted  to  lower  animals  by  use  of  these 
organisms. 

Dengue  Fever. — This  is  an  acute  infectious  disease  of  the 
tropics,  characterized  by  fever,  skin  eruptions,  rheumatoid  pains, 
an  afebrile  remission  and  a  febrile  end,  due  to  a  filterable  virus, 


SCARLET   FEVER  263 

transmitted  by  the  mosquito,  Culex  fagitans.  The  virus  is  in 
the  blood-stream.  One  attack  gives  immunity;  little  is  known 
of  the  virus. 

Three-day  or  Sand-fly  Fever. — A  mild  infectious  disease 
chiefly  of  southeastern  Europe,  due  to  a  virus  which  will  pass 
through  a  bacteria-proof  filter  and  is  transmitted  by  the  sand-fly, 
Phlebotomus  pappatacii.  Cultures  have  not  been  obtained. 

Typhus  Fever  or  Spotted  Fever. — An  acute  epidemic  disease 
with  prolonged  course,  prostration,  a  macular  eruption,  ending  by 
crisis,  transmitted  by  the  body  louse,  Pediculus  vestamenti.  The 
virus  is  filterable  but  is  obtained  with  difficulty.  It  is  found  best 
toward  the  end  of  the  fever.  It  may  be  transmitted  to  monkeys. 
It  is  destroyed  quickly  at  52°C.  Brill's  disease  is  a  mild  typhus 
fever.  A  Gram-positive  anaerobic  non-motile  bacillus  .2-.6  X 
.9-2/1,  has  been  described  as  inhabiting  the  blood  in  typhus.  As 
claims  to  being  the  cause  of  the  disease  one  finds  that  injection 
causes  a  distinct  febrile  reaction  in  guinea  pigs  and  the  culture 
may  be  used  as  antigen  in  a  complement  fixation  test. 

Poliomyelitis. — An  acute  infectious  disease,  chiefly  of  children 
characterized  by  a  short  febrile  attack,  followed  by  a  rapidly 
appearing  paralysis  in  various  muscles.  Means  of  transmission 
from  child  to  child  is  unknown,  but  it  has  lately  been  shown  that 
the  stable  fly,  Stomoxys  calcitrans,  can  transmit  it  from  monkey 
to  monkey.  Greater  weight  is,  however,  laid  upon  transmission 
from  person  to  person  by  contact  and  the  emanations  from  the 
upper  respiratory;  this  idea  gains  in  value  because  monkeys  can  be 
infected  by  nasal  washings  from  patients  during  the  disease.  The 
virus  is  in  the  central  nervous  system,  lymphatic  system,  blood, 
succus  entericus,  nasal  mucous  and  various  organs.  It  is  said  to 
be  constantly  in  the  nasal  mucosa  of  not  only  patients  but  of  the 
well  in  their  vicinity.  This  is  supposed  to  be  its  portal  of  entry  to 
the  body.  It  is  transmitted  to  monkeys  by  injecting  emulsions 
of  the  virus-containing  parts  into  the  brain,  blood-stream  or 
peritoneum.  It  can  be  filtered  through  porcelain.  It  resists 


264  THE  FILTERABLE  VIRUSES 

glycerine,  drying  and  autolysis.  It  is  destroyed  at  5o°C.  in  one- 
half  hour.  The  virus  can  be  cultivated  by  growing  bits  of  brain 
and  cord  from  a  case  dead  of  the  disease  in  unheated  ascitic  fluid 
to  which  bits  of  sterile  tissue  have  been  added;  the  growth  is 
anaerobic.  It  appears  as  tiny  globoid  bodies,  singly,  in  pairs  or 
short  chains.  Appropriate  staining  will  demonstrate  them  also 
in  the  nervous  tissues. 

Active  artificial  immunity  and  some  passive  immunity  have 
been  obtained  but  these  are  not  of  therapeutic  value.  One 
attack  probably  confers  immunity.  In  the  therapeutics  of  the 
disease  it  is  practicable  to  inject  into  the  spinal  column,  the  blood 
serum  of  human  cases  that  have  recovered  from  the  disease;  this 
indicates  that  virus-neutralizing  bodies  are  formed  during  an 
attack. 

Foot  and  Mouth  Disease. — An  acute  infectious  disease  of  cat- 
tle, characterized  by  a  vesicular  eruption  in  the  mouth  and  around 
the  crown  of  the  hoof.  It  may  be  transmitted  to  man  by  the 
use  of  milk  from  infected  cows.  It  is  also  directly  communicable. 
It  has  not  been  cultivated.  It  is  filterable;  it  is  said  to  be  due 
to  the  Cytorrycetes.  It  is  destroyed  at  5o°C.  in  ten  minutes, 
easily  by  freezing  and  ordinary  disinfectants.  One  attack  gives 
no  lasting  immunity  but  the  blood  is  said  to  contain  anti-bodies 
immediately  after  the  attack,  which  will  be  protective  to  other 
animals. 

Trachoma. — An  infectious  inflammation  of  the  conjunctiva 
with  the  production  of  minute  but  visible  nodules  on  the  under 
sides  of  the  lids.  By  some  it  is  said  to  be  due  to  a  form  of  the 
influenza  bacillus,  by  others  to  an  invisible  virus.  It  is  directly 
communicable,  filterable  and  transmissible  to  monkeys.  It  has 
not  been  cultivated. 

Measles. — An  acute  eruptive  fever  due  to  a  filterable  virus 
which  is  found  in  the  blood,  buccal  and  nasal  secretions.  It  is 
transmissible  to  monkeys  by  inoculations  of  patient's  blood,  even 
before  the  Koplik  spots  appear.  It  persists  in  the  blood  until 


MUMPS  265 

after  the  appearance  of  the  eruption.  It  resists  drying  and  freez- 
ing. It  is  destroyed  at  55°C,  in  fifteen  minutes;  it  has  not  been 
cultivated.  Immunity  follows  an  attack  but  no  passive  immunity 
has  been  reported. 

Mumps. — This  disease  seems  also  to  be  due  to  a  filterable  virus, 
resident  within  the  parotid  gland,  capable  of  transmission  to 
monkeys.  Many  organisms  have  been  described  but  none  is 
probably  specific. 

It  must  be  said  of  both  the  hypothetical  organisms  of  variola 
and  scarlatina,  that  if  they  are  the  cause  of  these  two  diseases 
they  differ  from  all  other  known  protozoan  parasites,  because 
the  latter  require  an  intermediate  host  for  the  transmission  of 
the  parasite  from  individual  to  individual  while  these  certainly 
do  not. 

Rocky  Mountain  Fever  is  not  due  to  a  filterable  virus  but  its 
agent  resides  in  the  blood.  It  is  a  disease  of  the  Rocky  Mountain 
States  characterized  by  general  pains;  macular  eruption  and  con- 
stitutional symptoms  of  infection,  it  is  transmitted  by  means  of  the 
tick,  Dermacentor  venustus.  The  female  tick  obtains  the  agent 
by  blood  sucking  and  can  transmit  it  to  her  young.  The  virus 
is  destroyed  by  heating  and  drying.  A  minute  coccoid  body  has 
been  found  in  the  tick  but  it  has  not  been  cultivated.  One 
artificial  attack  in  the  guinea  pig  leaves  immunity,  as  does  the 
spontaneous  disease  in  man. 

Trench  Fever  is  a  disease  which  became  known  in  the  Great 
War  characterized  by  fever,  pains  in  the  legs,  back  and  head,  with 
marked  dizziness,  due  to  a  resistant  filterable  virus,  in  all  proba- 
bility transmitted  by  lice;  the  virus  is  found  in  the  blood.  The 
entrance  is  effected  either  by  the  bite  of  a  louse  or  its  f  eces  may  be 
scratched  into  tiny  wounds  on  the  skin.  The  virus  seems  to  be 
excreted  in  the  urine  and  sputum.  It  is  not  killed  at  8o°C.  The 
louse  is  not  infective  for  seven  days  after  biting  a  patient  and 
then  remains  infective  for  three  weeks.  No  immunity  follows  an 
attack. 


266  TTHE  FILTERABLE  VIRUSES- 

Encephalitis  lethargica  is  a  disease  alleged  to  be  due  to  a 
virus.  It  appears  sometimes  in  epidemic  form  and  was  observed 
after  the  last  pandemic  of  influenza;  some  persons  believe  the  two 
are  related.  Some  students  of  the  subject  report  successful  trans- 
mission to  monkeys. 


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DESCRIPTION  OF  PLATE  I 
Malarial  Parasites 

i.  Two  tertian  parasites  about  thirty-six  hours  old,  within  blood  cor- 
puscles magnified. 

2  Tertian  parasite  about  thirty-six  hours  old;  stained  by  Romanowsky's 
method  The  black  granule  in  the  parasite  is  not  pigment  but  chromatin. 
Next  to  it  and  to  the  left  is  a  large  lymphocyte,  and  under  it  the  black  spot 
is  a  blood  plate. 

3.  Tertian  parasite  division  form  nearby  is  a  polynuclear  leucocyte. 

4.  Quartan  parasite  ribbon  form. 

5.  Quartan  parasite,  undergoing  division. 

6.  Tropical   fever  parasite    (aestivo-autumnal).     In  one  blood   corpuscle 
may  be  seen  a  smaller,  medium,  and  large  tropical  fever-ring  parasite. 

7.  Tropical  fever  parasite.     Gametes  half-moon  spherical  form.     Smear 
from  bone  marrow 

8.  Tropical  fever  parasite  which  is  preparing  for  division  heaped  up  in  the 
blood  capillaries  of  the  brain. 

Asexual  Forms 

9.  Smaller  tertian  ring  about  twelve  hours  old. 

10.  Tertian  parasite  about  thirty-six  hours  old,  so-called  amoeboid  form, 
n.  Tertian  parasite  still  showing  ring  form,  forty-two  hours  old. 

12.  Tertian   parasite,   two  hours  before   febrile  attack.     The  pigment  is 
beginning  to  arrange  itself  in  streaks  or  lines. 

13.  Tertian  parasite  further  advanced  in  division.     Pigment  collected  in 
large  quantities. 

14.  Further  advanced  in  the  division  (tertian  parasite). 


PLATE   I 


DESCRIPTION  OF  PLATE  II 
Malarial  Parasites 

15.  Complete  division  of  the  parasite.     Typical  mulberry  form. 

16.  To  the  left  is  the  completed  division  form,  an  almost  developed  gamete 
which  is  to  be  recognized  by  its  dispersed  pigment. 

17.  A  tertian  ring  parasite,  small  size  broken  up. 

1 8.  Three-fold  infection  with  tertian  parasite.     The  oval  black  granules 
are  the  chromatin  granules. 

19.  To  the  left,  tertian  parasite  with  large,  sharply  demarked,  and  deeply 
colored  chromatin  granules.     To  the  right,  tertian  parasite.     Both  thirty- 
six  hours  old.     Both  probably  gametes. 

20.  Tertian  parasite  thirty-six  hours  old,  ring  form. 

21.  Tertian  parasite  with  beginning  chromatin  division,  with  eight  chrom- 
atin segments. 

22.  Tertian  parasite  chromatin  division  farther  advanced  with  twelve 
chromatin  granules,  in  part  triangular  in  form. 

23.  Completed  division  figure  of  a  tertian  parasite.     Twenty-two  chrom- 
atin granules. 

24.  The  young  tertian  parasites  separating  themselves  from  each  other. 
The  pigment  remains  behind  in  the  middle. 

25.  Quartan  ring  parasite,  which  is  hard  to  differentiate  from  large  tropical 
ring  or  small  tertian  ring. 

26.  Quartan  ring  lengthening  itself. 

27.  Small  quartan  ribbon  form. 

28.  The  quartan  ribbon  increases  in  width.     The  dark  places  consist  almost 
entirely  of  pigment. 


PLATE  II 


\   A 


DESCRIPTION  OF  PLATE  HI 
Malarial  Parasite 

29,  30,  31.     The  quartan  ribbon  increases  in  width.     The  dark  places 
consist  almost  entirely  of  pigment. 

32.  Beginning  division  of  the  quartan  parasite  and  the  black  spot  in  the 
middle  is  the  collected  pigment. 

33.  Quartan  ring. 

34.  Double  infection  with  quartan  parasites. 

35.  Wide  quartan  band.     The  fine  black  stippling  in  the  upper  half  of  the 
parasite  is  pigment. 

36.  Beginning  division  of  the  quartan  parasite.     The  chromatin  (black 
fleck)  is  split  into  four  parts. 

37.  Division  advanced,  quartan  parasites. 

38.  Typical  division  figure  of  the  quartan  parasite. 

39.  Finished  division  of  the  quartan  parasite.     Ten  young  parasites,  pig- 
ment in  the  middle. 

40.  Young  parasites  separated  from  one  another/ 

41.  Small  and  medium  tropical  ring,  the  latter  in  a  transition  stage  to  a 
large  tropical  ring. 

42.  Small,  medium  and  large  tropical  ring,  together  in  one  corpuscle. 


PLATE  III 


DESCRIPTION  OF  PLATE  IV 
Malarial  Parasite 

43.  To  the  left  a  young  (spore)  tropical  parasite.     To  the  right  a  medium 
and  large  tropical  parasite. 

44.  An  almost  fully  developed  tropical  parasite.     The  black  granules  are 
pigment  heaps. 

45.  Young  parasites  separated  from  one  another.     Broken  up  division 
forms  twenty-one  new  parasites. 

46.  To   the  left   a   red  corpuscle   with  basophilic,   karyochromatophilic 
granules.     Prototype  of  malarial  parasite.     On  the  right  a  red  blood  corpuscle 
with  remains  of  nucleus. 

Sexual  Forms  or  Gametes 

47.  An  earlier  quartan  gamete  (macrogametocyte  in  sphere  form),  female. 

48.  An  earlier  quartan  gamete  (microgametocyte),  male. 

49.  Tertian  gamete,  male  form  (microgametocyte). 

50.  Tertian  gamete,  female  (macrogamete). 

51.  Tertian  gamete  (macrogametocyte  still  within  a  red  blood  corpuscle. 

52.  Microgamete  tertian  within  a  red  blood  corpuscle. 

53.  Tropical  fever.     (^Estivo-autumnal)  gamete,  half  moon  (crescent)  still 
lying  in  a  red  blood  corpuscle.     In  the  middle  is  the  pigment.     The  concave 
side  of  the  crescent  is  spanned  by  the  border  of  the  red  blood  corpuscle. 

54.  Gamete,  tropical  fever  parasite. 

55.  Gamete  of  tropical  fever  parasite  heavily  pigmented. 

56.  Gamete  of  the  tropical  fever  parasite  (flagellated  form),  microgameto- 
cyte sending  out  microgametes  (flagella  or  spermatozoon). 


PLATE  IV 


CHAPTER  XI 
BACTERIOLOGY  OF  WATER,  AIR,  SOIL,  AND  MILK 

Bacteriological  examination  of  water  is  of  importance  for  the 
determination  of  the  presence  of  pathogenic  bacteria,  and  for  the 
enumeration  of  the  total  number  of  all  bacteria  contained  therein, 
the  latter  being  considered  an  index  of  the  purity  of  the  water. 

Several  well-known  pathogenic  bacteria  have  been  found  in 
water;  among  these  are  the  typhoid,  anthrax,  cholera,  plague,  and 
colon  bacilli,  also  the  pus  cocci.  Since  the  tetanus  bacillus  is  a 
normal  inhabitant  of  the  cultivated  soil  and  manure,  it  is  not  at 
all  uncommon  to  find  it,  at  times,  in  muddy  waters. 

Bacteriological  examinations  of  water  are,  in  a  measure,  very 
disappointing,  because  it  is  difficulty  and  at  times  impossible  to 
determine  the  presence  of  the  typhoid  bacillus,  even  when  it  is 
certain  that  it  is  present;  having  been  added  to  water  to  be  ex- 
amined it  is  even  then  difficult  to  isolate. 

The  fact  that  the  colon  bacillus  is  always  found  in  water  con- 
taminated by  faeces  is  a  great  help  in  the  recognition  of  polluted 
water.  In  the  case  of  typhoid  contamination  the  typhoid  bacillus 
may  elude  detection,  but  the  colon  bacillus  is  easily  found;  we 
may  then  assume  that,  since  it  is  impossible  for  typhoid  bacilli 
to  reach  water  without  the  colon  bacilli  that  water  having  no 
colon  bacilli  is  also  free  from  typhoid  bacilli.  Also  water  having 
colon  bacilli  in  great  numbers  is  contaminated  with  faeces,  and 
perhaps  typhoid  faeces.  The  detection  of  the  colon  bacillus  is 
therefore  of  prime  importance  in  the  examination  of  drink- 
ing water.  Its  detection  is  simple.  Water  must  be  collected 
in  sterile  bottles,  using  every  precaution  against  accidental 
contamination.  Fermentation  tubes  are  employed,  containing 
bouillon  with  i  percent  of  lactose.  Into  a  series  of  these  tubes, 

278 


BACTERIOLOGY   OF   WATER  279 

varying  amounts  of  water  are  run  by  means  of  a  sterile  pipette, 
2  c.c.,  i  c.c.,  .5  c.c.,  .1  c.c.,  .01  c.c.,  of  water  being  used.  After  a 
stay  of  twenty-four  hours  in  the  incubator,  if  gas  appears,  the 
bouillon  should  be  examined  by  plate  cultures  for  the  colon  bac- 
illus. Lactose  litmus  agar  is  used,  and  where  colonies  appear  that 
redden  the  litmus  and  resemble  the  colon  colonies  in  appearance, 
they  are  planted  in  milk,  fermentation  tubes,  peptone  solution, 
neutral  red  agar,  nitrate  solution,  and  gelatine,  and  the  various 
reactions  in  the  various  media  noted.  Some  idea  of  the  numerical 
presence  of  colon  bacilli  can  also  be  obtained.  Definite  quanti- 
ties of  the  raw  water,  similar  to  those  used  in  the  fermentation 
tubes,  may  be  plated  directly  without  previous  incubation.  A 
deeply  tinted  litmus  lactose  agar  is  used  and  upon  this  medium 
colon  bacillus  colonies  appear,  small,  pink,  round  or  whetstone- 
shaped  surrounded  by  a  pink  zone  or  halo.  Such  pink  colonies 
are  fished  out  into  the  different  media  as  above.  If  there  were 
twenty  pink  colonies  of  the  colon  type  upon  a  plate  of  litmus 
lactose  agar  that  had  been  seeded  with  i  c.c.  of  water  and  of 
these  eight  were  fished  and  determined,  with  the  discovery  that 
four  only  were  true  B.  coli,  we  would  assume  that  in  i  c.c.  of 
raw  water  half  the  pink  growing  colonies  were  those  of  B.  coli 
and  that  the  water  contained  ten  B.  coli  per  cubic  centimeter. 

The  significance  of  the  colon  bacilli  is  often  overestimated. 
They  are  found  in  all  rivers,  and  often  reach  streams,  wells,  and 
even  springs  by  contamination  from  the  barnyard,  or  manured 
fields.  Attempts  to  separate  colon  bacilli  from  human  and  animal 
sources  have  been  unsuccessful.  Some  authorities  use  strepto- 
cocci of  the  faecal  type  as  pollution  indictors.  This  is  not  abso- 
lutely reliable. 

Typhoid  bacilli  have  been  found  in  water.  One  way  that  is 
sometimes  successful  is  to  take  25  c.c.  of  a  4  percent  peptone  solu- 
tion and  add  this  to  a  litre  of  the  water  to  be  examined;  from  this, 
after  twenty-four  hours  in  an  incubator,  plates  may  be  prepared 
with  the  agar  medium  of  Endo  as  already  given  on  page  120. 


280  BACTERIOLOGICAL   EXAMINATIONS 

To  Count  Bacteria  in  Water 

The  sample  must  be  collected  in  a  sterile  bottle,  and  the  plates 
poured  immediately,  since  bacteria  multiply  enormously  after  a 
few  hours. 

Take  ^fo  c.c.  or  J^j  c.c.  or  i  c.c.  of  the  water  in  sterile  pipettes 
and  mix  with  a  tube  of  melted  gelatine  or  agar,  pour  quickly  into 
cool  sterile  petri  dishes  and  place  in  a  cool  dry  place.  The  Ameri- 
can Public  Health  Association  also  recommends  the  use  of  +  i 
percent  agar  plates  grown  both  at  room  and  body  temperature. 
The  counts  for  the  two  are  averaged.  After  forty-eight  hours 
count  the  colonies  and  the  result  (after  multiplication  where  3^0 
or  J^  c.c.  of  water  was  used)  will  be  the  number  of  bacteria  per 
cubic  centimeter.  It  may  be  necessary  to  dilute  the  water  five 
or  ten  times  before  pouring  plates.  A  glass  plate  ruled  into 
squares,  known  as  a  WolrThiigel  plate,  should  be  used  for  counting. 
The  number  of  bacteria  in  potable  waters  varies  in  many  ways, 
according  to  the  amount  of  pollution,  or  albuminous  matter  in 
the  water,  while  depth,  and  the  swiftness  with  which  it  flows  are 
conditions  that  modify  bacterial  contents.  The  water  in  a 
reservoir  becomes  almost  free  from  bacteria  during  the  first  ten 
days.  The  number  of  bacteria  diminishes-  10  percent  per  day 
for  the  first  five  or  eight  days,  due  no  doubt  to  gravitation  of 
the  bacteria  to  the  bottom,  also  in  part  to  the  action  of  light, 
which  plays  an  important  role  in  the  destruction  of  the  bacteria 
of  water  supplies. 

In  general,  water  containing  less  than  100  bacteria  per  cubic 
centimeter  is  considered  to  be  from  a  deep  source,  and  uncon- 
taminated  by  drainage.  Deep  artesian  wells  often  contain  but 
from  5  to  15  bacteria  per  cubic  centimeter,  water  from  rivers 
often  contain  12,000  or  20,000  depending  somewhat  upon  the 
season  of  the  year.  Rains  cause  an  augmentation  of  the  bac- 
terial content.  Summer  causes  a  diminution. 

In  identifying  a  certain  water  supply  as  the  cause  of  an  epi- 


TO    COUNT  BACTERIA   IN   WATER  281 

demic  of  typhoid,  the  number  of  bacteria  is  of  great  value  in 
locating  the  place  of  infection. 

The  efficiency  of  niters  in  large  municipal  water  supplies  is 
known  only  by  the  bacterial  content  of  the  effluent.  In  good 
sand  and  mechanical  (alum)  niters,  the  reduction  in  the  number 
of  bacteria  is  often  over  95  percent  (sometimes  99  percent). 
Plate  cultures  should  be  made  daily  from  every  filter  in  order  to 
determine  how  each  filter  is  performing.  Sand  filters  should  not 
filter  more  than  1,000,000  gallons  per  acre  a  day.  They  should  be 
at  least  i  metre  thick;  the  upper  Y^  inch  of  the  sand  performs  over 
90  percent  of  the  filtration,  due  to  a  certain  zooglea,  or  growth 
of  bacteria.  Cracks,  or  imperfections  in  the  filter  beds  are 
quickly  detected  by  the  rapid  increase  of  the  number  of  the  bac- 
teria in  the  effluent.  It  is  supposed  that  not  only  are  bacteria 
filtered  by  the  sand  but  that  destructive  changes  occur  in  the 
filter  which  greatly  diminish  the  number  of  bacteria.  A  filter 
must  be  used  for  a  few  days  before  it  becomes  efficient  or  "ripe. " 
After  a  time  it  becomes  inefficient  and  it  must  then  be  scraped, 
finally  the  sand  must  be  removed  and  washed. 

A  sand  filter  is  a  highly  efficient  means  of  water  purification. 
It  often  converts  a  foul  dirty  water  into  a  bright,  clean,  whole- 
some water  of  low  bacterial  content. 

Mechanical  filters  depend  for  their  efficiency  upon  the  addition 
of  aluminum  sulphate  to  the  water.  This  is  decomposed  by  the 
carbonates  and  aluminum  hydroxide  is  produced,  which  is  a 
white  jelly-like  flocculent  precipitate,  which  mechanically  en- 
tangles bacteria  and  removes  them  from  the  water.  Mechanical 
filters,  as  a  rule,  are  highly  efficient.  Domestic  filters,  even  the 
Pasteur,  are  often  unreliable. 

In  time  of  epidemics  of  cholera  and  typhoid  even  filtered  water 
should  be  boiled  before  use,  as  it  was  found  by  experiments  in  the 
Medico-Chirurgical  Laboratories  that  typhoid  bacilli  live  longer 
in  filtered  water  than  in  bouillon;  they  may  even  live  three  months. 


282  BACTERIOLOGICAL   EXAMINATIONS 

The  fewer  the  number  of  other  bacteria  the  longer  will  typhoid 
live.  They  can  live  many  days  in  ordinary  river  water. 

Ice  may  contain  great  numbers  of  bacteria;  it  is  well  known 
that  freezing  does  not  destroy  pathogenic  bacteria,  such  as  the 
typhoid  bacillus.  Prudden  found  typhoid  bacilli  in  ice  after  one 
hundred  days,  although  the  number  was  greatly  reduced  over  that 
placed  in  the  ice  originally.  Many  are  squeezed  out  by  contraction 
of  the  water.  The  greatest  danger  from  ice  is  in  dirty  handling. 

Disposal  of  sewage  is  a  bacteriological  process  in  many  cases; 
either  the  sewage  may  be  treated  in  sand  niters  or  it  may  be  run 
put  on  land  where  over  200,000  gallons  may  be  disposed  of  on  an 
acre  of  land  a  day.  As  far  as  possible  nature  should  be  imitated 
in  every  way  and  the  breaking  up  of  masses  of  matter  in  sewage 
may  be  accomplished  in  the  septic-tank  process  in  which  active 
oxidization  of  the  matter  is  accomplished  by  bacteria.  It  appears 
from  the  observations  of  many  sanitarians  that  both  aerobic  and 
anaerobic  bacteria  are  necessary  to  finally  reduce  sewage  to  the 
elementary  gases  and  pure  water. 

In  the  interior  of  closed  tanks  and  in  the  depths  of  sand  niters 
anaerobic  conditions  prevail.  On  beds  of  coke,  and  on  the  sur- 
face of  sand  filters,  aerobic  conditions  obtain.  The  effluent  from 
a  septic-tank  sewage-disposal  plant  is  very  often  pure  water  from 
both  chemical  and  bacteriological  standpoints,  due  to  the  chem- 
ical action  of  the  bacteria. 

Bacteriology  of  the  Air 

That  the  lower  layers  of  the  earth's  atmosphere  contain  many 
bacteria  is  well  known.  The  air  over  the  sea  and  over  mountain 
ranges  is  freer  from  bacteria  than  the  air  over  arable  lands  and 
large  cities. 

When  air  is  still  and  confined,  all  bacteria,  according  to  Tyndall, 
gravitate  to  the  ground,  and  the  air  above  becomes  quite  sterile. 
The  atmosphere  of  sick  rooms,  hospitals,  public  conveyances, 
theatres,  etc.,  contains  many  bacteria  and  often  pathogenic  ones. 


BACTERIOLOGY   OF    THE   AIR  283 

The  pus  cocci,  tubercle  bacilli,  and  the  organisms  causing  small- 
pox, scarlet  fever,  and  measles,  all  may  contaminate  the  air. 

The  number  of  bacteria  in  a  given  quantity  of  air  may  be  accu- 
rately measured  by  means  of  a  Sedgwick-Tucker  aerobioscope; 
this  consists  of  a  large  cylindrical  glass  vessel  opening  at  either 
end  into  various  tubulations  (Fig.  82).  Into  one  of  these  granu- 
lated sugar  may  be  packed;  the  ends  are  then  plugged  with  cotton 
and  the  apparatus  sterilized.  To  examine  the  air,  a  litre  or  more 
is  drawn  through  the  sugar  and  the  latter  is  then  shaken  into  the 
large  cylinder  where  it  is  dissolved  in  melted  gelatine  culture 


FIG.  82. — Sedgwick-Tucker  aerobioscope.     (Williams.) 

media.  The  latter  is  distributed  over  the  interior  of  the  glass 
and  allowed  to  harden.  All  the  bacteria  that  were  in  a  litre  of 
air  having  been  mixed  with  gelatine  and  those  that  are  not  strict 
anaerobes  grow  in  the  gelatine  and  a  number  of  colonies  can 
then  be  counted. 

The  dust  of  dwellings  and  streets  contains  most  of  the  bacteria. 
Dried  sputum  is  ground  under  foot  and  swept  up  in  gusts  of  wind, 
and  the  contained  bacteria  are  thus  inhaled  and  do  harm.  The 
air  coming  quietly  from  the  lungs  is  pure  and  sterile.  Even  in 
active  disease  processes  of  the  throat  this  is  true.  In  case  the 
breath  comes  violently,  as  in  speaking,  coughing,  and  sneezing,  the 
reverse  is  the  case.  In  general  it  may  be  put  down  as  an  axiom 
that  disease  germs  cannot  rise  from  a  fluid,  such  as  sewage.  If 
they  could  it  would  mean  that  they  are  lighter  than  air,  which  is 
not  the  case.  Sewer  gas,  as  a  rule,  is  a  bearer  of  some  pathogenic 
bacteria  chiefly  cocci  but  in  reality  it  is  purer  than  generally  sup- 
posed. The  spread  of  organisms  from  sewage  only  extends  3-6 
metres  into  the  atmosphere  and  then  only  the  the  bursting  of  bub- 


284  BACTERIOLOGICAL   EXAMINATIONS 

bles  in  the  presence  of  gas  under  pressure;  air  currents  may  of 
course  carry  germs  so  freed  a  much  longer  distance. 

Bacteriology  of  the  Soil 

At  least  two  forms  of  pathogenic  bacteria  are  habitually  found 
in  the  soil.  The  tetanus  bacillus,  it  is  well  known,  exists  in  garden 
earth,  manure,  and  top  soil  generally.  Dirt  getting  into  wounds 
is  the  most  frequent  cause  of  tetanus.  Drinking  water  laden  with 
soil  has  been  known  to  have  in  it  tetanus  bacilli,  and  if  used  in 
an  unsterilized  condition  in  wounds  or  when  a  comparatively  feeble 
antiseptic,  such  as  creolin,  has  been  added,  it  may  cause  tetanus. 

The  gaseous  edema  group,  the  bacilli  of  malignant  edema, 
symptomatic  and  parasitic  anthrax  are  frequently  found  in  soil. 
The  highly  tilled  soil  of  the  battlefields  in  France  was  heavily 
laden  with  the  first,  hence  the  great  incidence  of  infection  after 
wounds  in  the  late  war.  Streptococci  and  colon  bacilli,  too,  have 
been  found  in  garden  soil.  Typhoid  bacilli  may  contaminate  soil, 
but  do  not  multiply  in  it.  In  sandy  soil  100,000  bacteria  per 
gram  have  been  found,  in  garden  soil  1,500,000  bacteria  per  gram, 
and  in  sewage-polluted  soil  115,000,000  bacteria  per  gram  have 
been  determined.  The  first  few  inches  of  ordinary  soil  contain 
most  of  the  bacteria,  after  a  depth  of  2  metres  no  bacteria  at  all 
are  found  and  the  earth  is  sterile. 

Soil  may  be  collected  in  sterile  sharp-pointed  iron  tubes,  and 
diluted  with  sterile  water  of  given  quantity  and  plates  poured 
from  it. 

Arable  lands  may  be  enriched  very  much  by  inoculating  them 
with 'certain  nitrifying  bacteria,  some  of  which  convert  ammonia 
into  nitrous  acid,  which  form  in  them  nitrites;  others  change 
nitrites  into  nitrates  (nitrosomonas) .  Certain  of  these  bacteria 
are  concerned  in  the  assimilation  of  nitrogen  from  the  atmosphere 
and  adding  to  the  nitrogen  content  of  the  soil,  thus  enriching  it. 
On  the  roots  of  some  plants,  alfalfa,  beans,  peas,  and  clover, 
minute  tubercles  develop.  These  little  growths  are  caused  by  the 


BACTERIOLOGY    OF    COW'S    MILK  285 

nitrifying  bacteria,  and  add  to  the  nutrition  of  the  plant  by  adding 
to  it  ammonia. 

Bacteriology  of  Cow's  Milk 

Theoretically  the  milk  in  the  interior  of  the  breasts  of  nursing 
women  and  the  udders  of  cows  is  sterile.  So  soon  as  it  leaves  the 
nipple  it  becomes  contaminated  with  bacteria,  and  by  the  time  it 
reaches  the  pail,  in  the  case  of  cow's  milk,  it  is  far  from  sterile. 

Bacteria  of  the  air,  and  dust  from  the  cattle  and  bedding,  at 
every  movement  of  the  cow,  and  by  the  agency  of  flies,  find  their 
way  into  milk  and  contaminate  it.  The  number  of  bacteria  that 
develops  in  the  milk  depends  upon  the  number  that  reach  it  in  the 
first  place,  the  temperature  of  the  air,  and  the  length  of  time  milk 
is  kept  at  a  temperature  favorable  for  their  multiplication.  Two 
hundred  and  thirty-nine  different  varieties  of  bacteria  have  been 
isolated  from  milk  at  different  times. 

Pathogenic  varieties  of  bacteria  that  have  been  found  in  cow's 
milk  include-  the  tubercle  bacillus,  Streptococcus  pyogenes,  Staphy- 
lococcus  aureus,  the  colon  bacillus,  typhoid  bacillus,  the  diphtheria 
bacillus,  and  a  whole  host  of  bacteria  that  sour  or  ferment  the 
milk  and  render  it  unwholesome  or  poisonous  for  young  children. 

Cattle  may  be  tuberculous,  and  the  tubercle  bacilli  may  reach 
the  milk  in  this  way.  There  may  be  abscesses  of  the  udder  and 
the  streptococci  from  the  pus  may  cause  infection  in  those  that 
use  it.  Ordinary  follicular  tonsillitis  may  be  caused  in  this  way. 

Bacteria  may  develop  rapidly  in  milk,  which  is  a  good  culture 
medium,  until  they  number  many  millions  per  cubic  centimeter 
(sometimes  200,000,000). 

In  good  milk  the  number  of  bacteria  may  increase  when  the 
temperature  is  go°F.,  from  5,200  originally  in  the  milk  imme- 
diately after  milking,  to  654,000  in  eight  hours. 

By  exposing  milk  to  a  temperature  of  i65°F.  for  twenty  to 
thirty  minutes  and  quickly  cooling  (Pasteurization)  most  of  the 
non-spore-bearing  bacteria  are  destroyed,  so  that  the  number  may 


286  BACTERIOLOGICAL   EXAMINATIONS 

be  reduced  99.999  percent  by  this  process.  The  Pasteurization 
of  milk  has  become  an  economic  problem  of  great  importance  in 
large  communities  and  is  not,  as  it  should  be,  sufficiently  super- 
vised. That  method  is  best  in  which  milk  is  held  at  i46°F.  for 
thirty  minutes.  No  harm  is  done  to  the  nutritional  value  of  the 
milk.  One  of  the  dangers  of  the  method  is  that  the  commercial 
Pasteurizing  machines  are  not  always  thoroughly  clean  and  them- 
selves contaminate  the  milk  when  discharging  it  after  heating. 
More  evidence  is  on  the  side  of  the  second  view.  The  practical 
importance  of  the  controversy  is  that  milk  whether  heated  or  not 
should  be  kept  at  a  temperature  at  which  bacteria  will  not  mul- 
tiply, under  6o°F.  Pasteurized  milk  is  safest  in  time  of  typhoid 
epidemics. 

Absolute  cleanliness  on  the  part  of  the  milker,  the  use  of  steril- 
ized gloves  and  clothes,  the  absence  of  flies,  dust,  and  the  imme- 
diate disposal  of  manure,  the  nitration  of  the  milk  after  collection, 
the  immediate  cooling  of  it,  the  uses  of  sterilized  milk  cans  and 
bottles,  all  lessen  the  bacterial  content  of  milk.  It  then  keeps 
better,  and  is  a  wholesomer  and  safer  food  for  infants,  especially 
in  hot  weather. 

By  drinking  water  containing  typhoid  bacilli  cows  cannot 
be  sources  of  typhoid  infection  through  trie  milk.  The  typhoid 
bacilli  are  not  transmitted  through  the  bodies  and  udders  of  the 
animals. 

A  bacteriologic  examination  of  milk  comprises  a  total  count, 
the  presence  of  colon  bacilli,  streptococci  in  pus  cells,  tubercle 
bacilli  and  special  species  as  the  case  suggests.  The  first  is  done 
as  given  for  water,  as  is  the  second.  The  discovery  of  streptococci 
is  made  by  centrifugalizing  a  definite  quantity  and  examining  the 
sediment  for  chains,  particularly  in  relation  to  leucocytes,  the  pus 
cells.  Tubercle  bacilli  are  found  by  injecting  guinea  pigs  or  by 
dissolving  the  milk  in  antiformin  (i  part  milk  and  i  part  15  per- 
cent antiformin)  warming  and  examining  the  sediment  after 
centrifugalization. 


INDEX 


Abscesses,  154 

Achorion  Schoenleinii,  231 

Acid,  benzoic,  56 

boric,  137 

fast,  1 06 

hydrochloric,  39,  137 

lactic,  167 

production,  128 
Acids.  137 

mineral,  137 
Acne,  154 

Acquired  immunity,  47 
Actinomyces,  3 

bovis,  224 

farcinicus,  223 

madura,  226 
Action,  hydrolytic,  25 
Active  immunity,  47 
Aedes  calopus,  259 
Aerobes,  19 
Aerobioscope,  283 
Aerogenes  mucosus,  166 
^Estivo-autumnal  parasites,  248 
Agar-agar,  117 
Agar,  blood,  119 

glycerine,  116 

Agglutinins,  53,  60,  92,  171,  172 
Aggressins,  44 
Air,  bacteria  of,  280 

borne  infection,  36 

liquid,  20 
Alcohol,  141 
Alexins,  51,  60 
Allergic,  63,  66 


Alternate  generation  245 
Amboceptor,  51.  60 
Ammonia,  129 
Amoeba  dysenterise,  32,  235 
Amoebae,  234 
Amoeboid  motion,  233 
Amphitrichous  bacteria,  211 
Anaerobes,  19 
Anaerobic  culture,  130 
Anaphylaxis,  63 
Andrade  indicator,  118 
Aniline  dyes,  97 
Animal  experiments,  132 

carriers,  37 

parasites,  232 

Anopheles  maculipennis,  249 
Anthrax  bacillus,  16,  31,  33,  50,  100, 

183 

anti-serum,  187 

vaccine,  83,  187 
Anti-aggressins,  45 
Antibiosis,  19 
Antibody,  49,  60 
Anti-complement,  61 
Anti-ferments,  60 
Antigens,  60,  61 
Anti-immune  body,  61 
Anti-leucocidin,  70 
Anti-plague  serum,  77,  83,  165 
Antisepsis,  135 

Antiseptic  values,  relative,  142 
Antiseptics,  135 
Anti-toxin  for  botulism,  70,  198 

for  diphtheria,  71,  194,  211 


287 


288 


INDEX 


Anti-toxin,  dosage,  74 

for  dysentery,  180 

for  plant  toxins,  70 

for  pyocyaneus,  70 

staphylococcus,  70,  154 
streptococcus,  75 

for   symptomatic   anthrax,    70, 
196 

for  tetanus,  70,  74,  192 

manufacture  of,  71 

standardization  of,  73 
Anti-toxins,  43,  54,  60,  70 

for  animal  toxins,  70 

standard,  73 
Arnold  sterilizer,  no 
Artesian  Wells,  280 
Arthrospores,  16 
Aspergillus,  5 

flavus,  231 

fumigatus,  231 

niger,  231 

Attenuation  of  bacteria,  34,  135 
Autoclav,  109 
Autopsies,  animal,  133 
Avenue  of  infection,  35 


Babes  Ernst  granules,  9 
tubercles,  258 

Bacillus,  2,  7 

aerogenes  capsulatus,  100,  199 
of  anthrax,  16,  31,  34,  50,  100, 

««3 

of  blue  pus,  181 

botulinus,  197 

Chauvoei,  194 

of  cholera,  202 

colon,  100,  174,  278,  284,  286 

comma,  202 

of  diphtheria,  31,  100,  207 

of  dysentery,  100,  178 


Bacillus,  enteriditis  sporogenes,  199 
Friedlander's,  166 
fusiformis,  201 
Gartner's,  177 
of  glanders,  31,  206 
Koch  Weeks,  162 
lepra,  31,  221 
of  lockjaw,  1 88 
malignant  oedema,  31,  192,  264, 

284 

mallei,  31,  100,  206 
of  Malta  fever,  31,  158 
Morax  and  Axenfeld,  162 
perfringens,  199 
of  plague,  31,  162 
pseudo-diphtheria,  213 
pyocyaneus,  100,  181 
rauschbrand,  194 
smegma,  219 
of  soft  chancre,  183 
of  symptomatic  anthrax,  194 
of  tetanus,  31,  74,  100,  188,  284 
of  tuberculosis,  31,  33,  98,  100, 

213, 286 
typhosus,  31,  100,  168,  278,  279, 

285 

Xerosis,  213 

Bacteria,  attenuation  of,  34 
of  air,  282 
biological  conditions  of  growth, 

18 

chemical  composition  of,  17 
chromogenic,  23 
definition  of,  i 
disposal  of,  32 
fixed  strains  of,  35 
higher,  4,  5,  17 
increasing  malignancy  of,  35 
lophotrichous,  n 
measuring  of,  9 
mesophilic,  19 


INDEX 


289 


Bacteria  of  milk,  285 

of  mouth,  38 

parasitic,  31 

photogenic,  23 

psychrophilic,  19 

reproduction  of,  1 2 

of  skin,  38 

of  soil,  284 

staining  of,  96 

of  stomach,  38 

study  of,  95,  120 

submicroscopic,  255 

thermophilic,  19 
Bacteriaceae,  2 
Bacterial  energy,  23 

proteins,  28 
Bacterins,  77,  140-154 

sensitized,  77 

Bacteriological  diagnosis,  108,  123 
Bacteriolysins,  10,  41,  49,  57,  60 
Bacteriolysis,  61 
Bacterium;  2 

aerogenes,  166 

Bulgaricum,  167 

coli,  174 

enteriditis,  177 

influenzas,  100,  154 

lactis  aerogenes,  166 

mucosus,  1 66 

pestis,  100,  162 

pneumonias,  166 

ulceris  chancrosi,  183 
Balantidium,  233 
Beggiatoa,  4 
Beggiatoaceae,  4 
Benzoate  of  soda.  56 
Benzoic  acid,  56 
Benzol  ring,  51,  55 
Biological   conditions  of  growth   of 

bacteria,  18 
Bismarck  brown,  99 
19 


Black-leg  vaccine,  84 
Blastomycetes,  5,  17,  228 
Blastomycosis,    228 
Blood  agar,  119 

cultures,  170 

serum,  112,  119 
Blue,  methylene,  98 

pus  bacillus,  181 
Boils,  154 

Bordet-Gengou   bacillus   of    whoop- 
ing-cough, 161 
Botulism,  197 
Bouillon,  113,  198 
Bovine  tuberculosis,  219 
Bromine,  137 
Bronchitis,  160 
Brownian  motion,  12,  95 

Capsule  staining,  101 
Capsules,  10,  15,  16 
Carbol  fuchsin,  98 

thionin.  100 
Carbolic  acid,  139 
Carbuncles,  154 
Carriers,  37,  38,  151.  170 
Cell  division,  13 
Cellulo-humeral  theory,  50 
Centrosome,  238 
Cercomonas,  233 
Chain  coccus,  143 
Chauvoei,  bacillus  of,  194 
Chemotaxis,  20,44,  46,   47,  50 
Chlamydobacteriaceae,  3,  8 
Chlamydozoa,  255 
Chloramin.  138 
Chloride  of  lime,  138 

of  zinc,  141 
Chlorinated  lime,  138 
Chlorine,  137 
Cholera  bacillus,  202 
Cholera,  vaccination  against,  79 


290 


INDEX 


Chromogenic  bacteria,  23 
Ciliata,  233 
Cladothrix,  3 
Classification,  i,  5 

CO,,.  23 

Coccaceae,  i 

Cocci,  5 

Coccidia,  233,  235,  253 

Coccidioides,  5 

immitis,  230 
Coccidiosis,  230 
Coccidum  hominis,  253 
Coccus  chain,  143 
Coccus,  Malta  fever,  158 

of  meningitis,  149 
Cold,  influence  of,  20 
Coley's  fluid,  88 
Collodion  sac,  113 
Colon  bacillus,  90,  174,  279,  286 
Comma  bacillus,  202 
Complement,  49,  51,  56,  57,  60,  61 
deviation,  69 
fixation,  67 
Complementophile,  61 
Conjunctivitis,  146,  156.  162 
Copper  sulphate,  137 
Copula,  60 
Corenybacterium  diphtheriae,  207 

pseudo-diphtherias,  273 
Counting  bacteria,  280 
Crenothrix,  4 
Creolin,  139 
Cresol/ 139 

Culture  media,  19,  102,  113 
Cultures,  120 

anaerobic,  130 
plate,  123 

Cyclasterion  Scarlatinale,  262 
Cytase,  49,  60 
Cytolysins,  53,  60 
Cytolysis,  53,  60 


Cytophile,  61 
Cytoplasm,  9 
Cytoryctes  variolae,  7} 
Cytotoxins,  60 


261 


Dakin's  solution,  138 

Dark  field  illumination,  106 

Darkness,  influence  of,  20 

Dengue  fever,  262 

Desensitization,  65 

Desmon,  60 

Diarrhoea,  170 

Dichloramin  T,  138 

Differentiation  of  B.  typhosus  and 

B.  coli,  176 
Dilution  method,  122 
Diphtheria,  144,  207 

anti-toxin,  54,  70,  192 

bacillus,  207 

stain,  105 

toxin,  43,  71,  210 

toxin-antitoxin  injections,  82 

virulence  test,  212 
Diplococcus,  5 

gonorrhoea,  155 

lanceolatus,  146 

meningitis,  100,  149 
Direct  division,  13 
Disinfectants,  135 
Disinfection,  135 
Dum-dum  fever,  240 
Dust,  36 
Dyes,  aniline,  97 
Dysentery,  amoeba,  234 

bacillus,  178 

Ectosarc,  234 
Egg  cultures,  120 
Ehrlich's  theory,  50 
Encephalitis  lethargica,  266 
Endo  medium,  120 


INDEX 


2QI 


Endocarditis,  144,  146,  156 

Endosarc,  234 

Endospores,  13 

Endotoxins,  28,  41,  50,  56,  57,  60 

Entamceba  coli,  235 

buccalis,  236 

histolytica,  234 

tetragena,  219,  236 
Enzymes,  24 
Erysipelas,  144 
Exhaustion  theory,  47 
Experiments,  animal,  132 

Farcin  du  Boeuf,  223 
Favus,  231 
Fermentation,  25 

tubes,  128 
Ferments,  24,  60 

diastatic,  24 

tryptic,  24 
Filters,  32,  101,  113,  263,  281 

alum,  281 

Pasteur,  113 

sand,  281 
Fixateur,  60 
Fixation,  96 
Flagella,  10,  12 

staining,  102 
Flagellata,  219,  233,  237 
Focal  infection,  41 
Fomites,  259 

Foot  and  mouth  disease,  264 
Formaldehyde,  139 
Fractional  sterilization,  no 
Frambcesia,  243 
Friedberger's  theory,  65 
Friedlander's  bacillus,  166 
Fuchsin  solutions,  98 

Gabbet's  solution,  106 
Ganglia,  259 
Gartner's  bacillus,  177 


Gaseous  edema  bacillus,  199 

Gastric  juice,  39 

Gelatine,  116 

Generation,  alternate,  215,  232,  241, 

259 

Giemsa's  stain,  101 
Glanders  bacillus",  206 
Glossina  palpalis,  239 
Glyco-nucleo-protein,  18 
Gonidia,  12,  17 
Gonococcus,  155 
Gonorrhoea,  156 
Gram's  method  of  staining,  99 
Granules,  chromophilic,  9 

Babes  Ernst,  9 
Gregarines,  233 
Gregarinida,  233 
Gruber-Dunham   reaction,   96,    155, 

171 
Gymnobacteria,  10 

H,  23 
H2S,  24 

Haemolysins,  60,  92 
Haemolysis,  52,  63 
Haemolytic  serum,  51,  53 
Haemosporidia,  228,  233,  245 
Haffkine,  79 
Halogens,  137 
Hanging  drop,  96 
Haptophores,  57,  6 1 
Hepatotoxin,  60 
Hemoglobinophilic,  161 
Heterotrichida,  233 
Hiss'  capsule  stain,  102 
Histological  methods,  133 
Human  tubercle  bacilli,  219 

transmission,  39,  218 
Hydrogen  ion  concentration,  114 

media  method,  114 

peroxide,  139 


INDEX 


Hydrochloric  acid,  36,  137 
Hydrophobia,  256 
Hyphomycetes,  5,  17,  230 
Hypersensitivity,  66 
Hypersusceptibility,  63 

Ice,  bacteria  in,  282 
Immune  body,  51,  59,  60,  61,  63 
Immunkorper,  60 
Immunity,  46 

acquired,  46 

active,  46 

anti-bacterial,  46 

anti-toxic,  46 

inherited,  47 

local,  38 

natural,  46 

passive,  46 

racial,  46 
Incubator,  in 
Index,  opsonic,  89 
Indicators,  115,  118 

Andrade,  118 
Indol  production,  129 
Infection,  30 

focal,  41 

mixed,  35,  50, 

phlogistic,  40 

secondary,  35 

septic,  40 

terminal,  39 

toxic,  40 
Infestation,  30 
Influenza  bacillus,  159 
Infusoria,  233 
Inoculating  animals,  132 

media,  121 
Insects,  37 

Intermediary  bodies,  60 
Involution  form,  8 
Iodine,  137 


Isoagglutination,  92 
Isohemolysin,  92 

Jenner,  78 
Jenner's  stain,  101 

Kidneys,  excretion  of  bacteria  by,  33 
Klebs-Loffler  bacillus,  207 
Koch's  postulates,  31 

Laboratory  technique,  108 
Lactic  acid,  25,  167 
Larva  of  mosquitos,  251 
Lateral  chain  theory,  50,  55 
Law  of  multiples,  55 
Leishman-Donovan  bodies,  240 
Leishman's  stain,  101 
Lepra  bacillus,  221 
Leucocytosis,  49 
Leutin,  28,  91,  241 
Lightning  rod  theory,  58 
Lime,  140 

chlorinated,  138 
Litmus  milk,  117 

tincture,  120 
Local  immunity,  38 
Lockjaw  bacillus,  188 
Loffler's  blood  serum,  119 

blue,  98 

flagella  stain,  104 

method  of  staining  tissues,  134 
Lophotrichous  bacteria,  n 
Lysis,  52 
Lysol,  139 

Macrogametes,  247,  249,  251 

Macrophages,  49 

Madura  foot,  226 

Malarial  parasites,  244 

Malignant  oedema,  bacillus  of,  192 

Mallein,  28,  86 

Malta  fever,  bacillus  of,  31,  158 


INDEX 


293 


Mannaberg's  scheme,  246 

Mastigophora,  233 

Measles,  264 

Measuring  bacteria,  9 

Meat  poisoning  bacillus,  197 

Membrane,  false,  26 

Meningitis,  144,  148,  149,  160,  182 

anti-serum,  76,  151,  161 
Meningococcus,  149 
Mercury  salts,  136 
Merismopedia,  2 
Merizoites,  247 
Mesophilic  bacteria,  19 
Metals,  influence  of,  21 
Micrococcus,  2,  6 

catarrhalis,  100,  151 

epidermidis  albus,  155 

gonorrhoea,  100,  155 

melitensis,  158 

pyogenes,  152 

tetragenus,  157 
Microgametes,  247,  249 
Microgametocytes,  247,  249 
Microphages,  49 
Microspira,  2 
Microsporon,  5 

furfur,  231 
Milk,  bacteria  of,  285 

borne  infection,  37 

litmus,  117 
Mixed  infection,  35 
Molecule,  toxin,  56 
Monadida,  233 
Monilia,  5 

Monkey  injection,  262,  263 
Monotrichous  bacteria,  n 
Mordants,  98 
Mosquitos,  249,  259 

aedes,  259 

anopheles,  249 

larva,  251 


Moulds,  5,  17,  230 
Muir-Pitfield  flagella  stain,  103 
Multiplication  of  bacteria,  1 2 
Mycelia,  5,  17 
Mycobacteriaceae,  3 
Mycobacterium,  3,  7 

lepra,  221 

tuberculosis,  213 
Mycomycetes,  5 
Mycoprotein,  18 

Needles,  inoculating,  123 

Negri  bodies,  257 

Neisser's  stain,  105 

Nephrotoxin,  60 

Neutralization  of  media,  114-116 

Nitrates,  129 

Nitrifying  bacteria,  24,  284 

Nitrites,  129 

reduction,  24 
Nitrogen,  24 
Nocardia,  222 
Novy  jars,  131 
Nutriment  of  bacteria,  19 

Oidia,  5 

Oidium  albicans,  228 
coccidoides,  228 
Oidiumycosis,  210,  228 
Oocysts,  251 
Ookinets,  251 
Opsonic  index,  89 
Opsonins,  60.  89 
Organelles,  235 
Osteomyelitis,  144,  154 

Paracolon  bacillus,  173,  177 
Paratyphoid  bacillus,  173 
Parasites,  animal,  30,  232 
Park  diphtheria,  treatment,  74 
Pasteur  filter,  112 


294 


INDEX 


Pasteurization  of  milk,  285 

Pathogenicity,  33 

Pathogens,  25 

Peptone  solution  (Dunham's),  118 

Pericarditis,  148 

Peritonitis,  144,  148 

Peritrichous  bacteria,  n 

Peroxide  of  hydrogen,  139 

Petrie  dishes,  123 

Pfeiffer's  reaction,  51 

Phagocytes,  47,  89 

Phagocytosis,  44,  47,  82,  89 

Phagolysis,  49 

Phlogistic  infection,  40 

Photogenic  bacteria,  23 

Phragmidothrix,  4 

Phycomycetes,  5 

Pink  eye,  162 

Pitfield's  flagella  stain,  103 

Pityriasis  versicolor,  231 

Placenta,  infection  through,  39,  218 

Plague  bacillus,  162 

vaccination,  83,  165 
Planococcus,  r,  6 
Planosarcina,  i,  6 
Plasmins,  27 
Plasmodium  falciparum,  248 

malarice,  246 

vivax,  247 
Pleomorphism,  8 
Pleuritis,  148,  154 
Pneumococcus,  146 

types,  76 

vaccination,  82 

Pneumonia,  144,  148,  154,  160,  167 
Poliomyelitis,.  263 

virus  of,  263 
Polymastigida,  233 
Polymites,  248 
Porcelain  filter,  112 
Postulates,  Koch's,  31 


Potassium  permanganate,  140 
Potato,  117 
Pragmidiothrix,  4 
Precipitins,  54,  60 
Preparateur,  60 
Proteins,  bacterial,  28 
Protozoa,  232 

staining  of,  106 
Pseudomonas,  2 
Psychrophilic  bacteria,  19 
Ptomaines,  25,  41 
Puerperal  fever,  144,  154 
Pus,  26,  40 
Putrefaction,  24 
Pyocyaneus,  anti-toxin,  70 

bacillus,  181 

Quartan  malarial  parasite,  246 

Racial  immunity,  46 
Rat  bite  fever,  243 
Rauschbrand  bacillus,  194 
Ravenel  potato  cutter,  117 
Ray  fungus,  224 
Reactivation,  51,  55 
Receptors,  57,  63 
Relapsing  fever,  243 
Retention  theory,  47 
Rheumatic  tetanus,  191 
Rhizopoda,  233 
Ringworm,  231 
Rocky  mountain  fever,  265 
Rod  bacteria,  2 
Roll  culture,  126 
Romano wsky's  stain,  101 
Roux  regulator,  112 
Russell's  medium,  120 

Sac,  collodion,  113 
Saccharomyces,  5 


INDEX 


295 


Saccharomycetes,  Busse,  230 

Sand-fly  fever,  263 

Sapraemia,  30 

Saprogens,  25 

Saprophytes,  18 

Sarcina,  2,  6 

Sarcode,  233 

Sarcodina,  233 

Scarlatina,  144,  262 

Scarlet  fever,  262 

Schizomycetes,  i 

Schizogony,  234,  246,  247,  251 

Secondary  infections,  35,  144,  154 

Septic  infections,  40 

tank,  282 
Septicaemia,  144,  148,  154,  162 

pneumococci,  148 
Serum,  anti-plague,  77,  165 

anti-pneumococcus,  75 

anti-toxic,  70 

haemolytic,  52 

reactivated,  51,  55 

shock,  66 

water,  119 

Sessile  phagocytes,  48 
Sewage  disposal,  282 
Silver  salts,  137 
Skin,  disinfection  of,  141 
Sleeping  sickness,  237 
Small  pox,  78,  154,  260 
Smegma  bacillus,  219 
Soft  chancre  bacillus,  183 
Soil,  bacteria  in,  284 

borne  infection,  37 
Soor,  228 
Sparing  action,  25 
Spermo toxin,  60 
Spirillaceae,  2,  202 
Spirillum,  2,  7 

cholera,  100,  202 
Spirochaeta,  3,  7,  233 


Spirochaeta,  carteri,  243 

duttoni,  243 

icterohemorrhagicae,  243 

morsus  muris,  243 

muris  ratti,  243 

nodosa,  243 

Novi,  243 

obermeieri,  243 

pallida,  240 

refringens,  240 

vincenti,  201 
Spirosoma,  2 
Sporangia,  17 
Spore  staining,  102 
Spores,  13,  no 
Sporoblasts,  251 
Sporogony,  234,  246,  2,51 
Sporothrices,  5,  249 
Sporothrix  schenki,  249 
Sporozoa,  233,  245 
Sporozoites,  246,  251 
Sporulation,  13,  96 
Spotted  fever,  263 
Stain,  Bismarck  brown,  99 

Fuchsin  solution,  98 

Giemsa's,  101 

Gram's,  99 

Hiss'  capsule,  102 

Leishman's,  101 

Loffler's  methylene  blue,  98- 
flagella,  104 

Neisser's  diphtheria,  105 

Pitfield's  flagella,  104 
modified  by  Muir,  103 

spore,  102 

thionin  blue,  100 

tubercle  bacilli,  106 

Weigert's,  134 

Welsh's  capsule,  101 

Wright's,  100 

Zeihl's  carbol-fuchsin,  98 


296 


INDEX 


Staining  bacteria,  96 
Standardization  of  anti-toxins,  73 

of  media,  114 
Staphylococcus,  2,  6 

albus,  152 

aureus,  152 

citreus,  152 

pyogenes,  152 
Stegomyia  calopus,  259 

Fasciata,  251 
Sterilization,  108,  135 

culture  media,  109 

fractional,  no 

glassware,  108 
Sterilizer,  Arnold,  no 
Sterling's  solution,  99 
Stern's  method  for  spirochetes,  106 
Stomach,  bacteria  of,  39 
Street  virus,  256 
Streptococcus,  2,  34,  143,  286 

antiserum,  75 

intracellularis,  149 

lanceolatus,  146 

mucosus,  149 

pneumonias,  100,  146 

pyogenes,  100,  143 

viridans,  146 
Streptothrix,  3 

hominis,  222 

madura,  226 
Study  of  bacteria,  120 
Substance  sensibilisatrice,  60 
Sulphur  dioxide,  140 
Symbiosis,  19 

Symptomatic     anthrax     anti-toxin, 
196. 

bacillus,  177,  193 
Syphilis,  241 

Table  of  characteristics  of  bacteria, 
267-269 


Temperature,   influence  on  growth, 

19 

Terminal  infection,  39 
Tertian  fever,  247 
Test,  tuberculin,  85,  91 

Schick,  91 

Tetanolysin,  44,  189 
Tetanospasmin,  44,  189 
Tetanus  anti-toxin,  74,  192 

bacillus,  31,  100,  188,  284 

rheumatic,  191 

spore,  1 88 

toxin,  28,  43,  44,  189 
Tetrads,  5 

Theory,  cellulo-humeral,  50 
Thermolabile,  51 
Thermostat,  in 
Thionin,  100 
Thiothrix,  4 

Thrombosis  formation.  26 
Thrush,  228 
Tonsillitis,  144 
Toxalbumins,  42 
Toxic  infection,  40 
Toxin,  28,  43,  60 

molecule,  55 
Toxoid,  43,  58 
Toxons,  43 
Toxophores,  57,  6 1 
Trachoma,  264 
Trench  fever,  265 

mouth,  202 
Treponema,  233 

pallidum,  91,  240 

pertenue,  243 
Trichobacteria,  n 
Trichomonas,  233,  240 
Trichomyces,  222 
Trichophyton,  5,  230 
Trypanosoma,  233,  237 

brucei,  237 


INDEX 


297 


Trypanosoma,  cruzi,  238 

equiperdum,  220,  238 

evansii,  237 

gambiense,  237,  239 

lewisi,  238 

noctuae,  220,  238 
Tsetse  fly,  237,  239 
Tubercle  bacillus,  213 

stain,  106 

Tubercles,  Babes,  258 
Tuberculin,  28,  85,  91,  220 

T.R.,  85 
Turpentine,  140 
Tyndallization,  no 
Typhoid  bacilli,  32,  35,  100,  168,  279 
284 

in  water,  278 

vaccination  against,  80 
Typhus  fever,  263 

Udder,  infection  by,  285 
Unit  of  anti-toxin,  73 

toxin,  73 
Uterus,  bacteria  in  normal,  39 

Vaccination,  77 

diphtheria,  82 

for  plague,  83 
Vaccine,  anthrax,  83 

black  leg,  84 

cholera,  79 

diphtheria,  82 

paratyphoid,  80 

plague,  83 

pneumonia,  82 

small  pox,  78 

tuberculosis,  85 

typhoid,  80 
Vaccinia,  78,  260 
Vaccinoid,  79 
Vacuoles.  10 


Variola,  78,  260 
Venom,  57 
Vibrio,  2,  7 

cholera,  202 

Metchnikovii,  206 

proteus,  206 

Schuylkilliensis,  206 

septique,  192 

tyrogenum,  206 
Vincent's  angina,  201 
Virulence,  33 
Virus  fixe,  257 

Wassermann's  list  of  anti-toxins,  64 

test,  70 
Water,  bacteria  of,  278 

borne  infection,  36 
Weigert's  aniline  gentian  violet,  99 

method  of  staining  tissue,  134 

theory,  57 
Weil's  disease,  243 
Welch's  capsule  stain,  101 
Wells,  artesian,  280 
Widal  reaction,  53,  171 
Wolffhiigel  plate,  280 
Wright's  stain,  100 

Xerosis  bacilli,  213 

Yaws,  243 
Yeasts,  5,  17,  228 
Yellow  fever,  258 

Zeihl's  solution,  98 
Zinc  chloride,  141 
Zooglea,  10 
Zwischenkorper,  60 
Zymase,  24 

Zymogenic  bacteria,  23 
Zymophore,  61 


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